the photosynthetic membrane: molecular mechanisms and biophysics of light harvesting

The Photosynthetic Membrane

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The Photosynthetic Membrane

Molecular Mechanisms and Biophysics of Light Harvesting

ALEXANDER RUBAN

School of Biological and Chemical Sciences, Queen Mary, University of London, UK

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This edition first published 2013

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Library of Congress Cataloguing-in-Publication Data

Ruban, Alexander (Alexander V.)

The photosynthetic membrane : molecular mechanisms and biophysics of light harvesting / Alexander Ruban.

p. cm.

Includes index.

ISBN 978-1-119-96054-6 (cloth) – ISBN 978-1-119-96053-9 (pbk.)

1. Photosynthesis. 2. Photosynthetic pigments. 3. Light absorption. I. Title.

QK882.R83 2013

572′.46–dc23

2012025765

A catalogue record for this book is available from the British Library.

Set in 10/12pt Times by SPi Publisher Services, Pondicherry, India

1 2013

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Dedicated to my family

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Contents

Preface xi Acknowledgements xiii

1 Life, Energy and Light 1 1.1 The Definition of Life 1

1.2 The Energy of Matter 2

1.2.1 The Source of Life’s Energy 3

1.3 Energy for the Future 3

1.4 Photosynthesis by Life 4

1.4.1 Photon Energy Transformations 5

Reference 6 Bibliography 6

2 The Space of the Cell 7 2.1 The Cell Concept: Fundamental Nature of Life 7

2.2 Compartmentalization: The Cult of the Membrane 9

2.3 Membrane Components: Fundamentals of Proteins 12

2.4 Functional Classification of Membrane Proteins 15


3 The Photosynthetic Membrane: Outlook 17 3.1 Knowledge of the Pre-Atomic Structure Era: Organization

of the Photosynthetic Membrane System 17

3.2 Composition of the Photosynthetic Membrane 21

3.2.1 Lipids 21

3.2.2 Lipid-Related Compounds of the Photosynthetic Membrane 22

3.2.3 Proteins and Protein Complexes 25

3.3 Oligomerization, Interactions and Mobility of the Photosynthetic

Proteins: Enabling Functions and Adaptations 28

3.3.1 Oligomerization and Clustering of Photosynthetic Membrane Proteins 28

3.3.2 Protein Mobility 30


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viii Contents

4 Popular Methods and Approaches to Study Composition, Structure and Functions of the Photosynthetic Membrane 33 4.1 Biochemistry and Molecular Biology Approaches 33

4.1.1 Isolation of Chloroplasts and Subchloroplast Particles 33

4.1.2 Isolation of Membrane Protein Complexes 35

4.1.3 Analysis of Lipids and Pigments 37

4.1.4 Protein Expression and Reconstitution In Vitro 38

4.1.5 Reconstitution of Membrane Proteins in Liposomes 39

4.1.6 Mutagenesis and Transgenic Manipulations 40

4.2 Visualization Techniques 41

4.2.1 Optical Microscopy 41

4.2.2 Electron Microscopy (EM) 42

4.2.3 Atomic Force Microscopy (AFM) 45

4.2.4 Crystallography Methods 45

4.3 Function Probing Methods 48

4.3.1 Absorption-Based Approaches 49

4.3.2 Raman Spectroscopy 54

4.3.3 Fluorescence-Based Approaches 55

References 65 Bibliography 65

5 Primary Processes of the Light Phase of Photosynthesis: Principles of Light Harvesting in Antennae 67 5.1 The Nature of Light 67

5.2 Absorption of Light by Molecules 71

5.3 Fate of Absorbed Light Energy 73

5.4 The Need for the Photosynthetic Antenna and the Fifth

Fate of Excitation Energy 75

5.5 Photosynthetic Antenna Pigments 81

5.5.1 Chlorophylls 82

5.5.2 Xanthophylls 87

5.6 Variety and Classification of Photosynthetic Antennae 91

5.7 Principles of Light Harvesting: Summary 93

5.8 Connecting Light Harvesting Antenna to the Photosystems:

Red Energy Traps 96


6 Towards the Atomic Resolution Structure of Light Harvesting Antennae: On the Path of Discoveries 101 6.1 Discovery and Primary Characterization of the Higher

Plant Antenna Complex 102

6.2 Development of Isolation Methods: Intactness,

Purity and Quantity 104

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Contents ix

6.3 LHCII Crystallography: The Beginnings 107

6.4 Revealing the Atomic Resolution Structure of LHCII

Antenna Complexes 111

6.4.1 Key Biochemical and Spectroscopic Advances that Aided the Emergence of the Current Atomic LHCIIb Structure 111

6.4.2 The New Structure of LHCIIb 115

6.5 Structure of a Minor LHCII Complex CP29 126

6.6 Comparison of LHCII Structure with the Structure of a Simpler

Light Harvesting Complex from Purple Bacteria, LH2 129


7 Structural Integration of Antennae within Photosystems 135 7.1 Light Harvesting Complexes Gene Family 136

7.2 Toward the Structure of a Complete Photosystem II Unit:

Supercomplexes 137

7.3 Supramolecular Structure of Photosystem I: LHCI 145

7.4 Photosynthetic Membrane Protein Landscapes 147

7.5 Robustness of the Light Harvesting Antenna Design: Resurrecting

the Structure to Preserve the Function 150


8 Dynamics of Light Harvesting Antenna: Spectroscopic Insights 159 8.1 Steady-State Optical Spectroscopy of LHCII:

Composition and Order 160

8.2 Time-Resolved Spectroscopy of LHCII: Energy Migration 165

8.2.1 Time-Resolved Fluorescence Spectroscopy 165

8.2.2 Time-Resolved Absorption Spectroscopy 167

8.3 Spectral and Structural Identity of LHCII Xanthophylls 170

8.4 Plasticity of Light Harvesting Antenna Design: Tailoring the

Structure to Optimize the Function 176

8.5 LHCII Oligomerization: Dynamics of the ‘Programmed Solvent’ 179

8.5.1 Alterations in the Spectral Properties of LHCII 179

8.5.2 Structural Changes within LHCII 183

8.6 Kinetics of the Collective LHCII Transition into the Dissipative

State: Exploring ‘The Switch’ Control 189


9 Adaptations of the Photosynthetic Membrane to Light 197 9.1 The Need for Light Adaptations and their Various Strategies 198

9.2 Long-Term Regulation of the Photosystem Ratio and their

Antenna Size: Acclimation 201

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x Contents

9.3 Short-Term Adaptations to Light Quality: State Transitions 202

9.3.1 The Phenomenology of State Transitions 202

9.3.2 The Molecular Mechanism of State Transitions 205

9.3.3 Chromatic Adaptations in Plants Lacking the Polypeptides of the Major LHC II Complex 209

9.3.4 Future of State Transitions Research 212

9.4 Short-Term Adaptations to Light Quantity 214

9.4.1 Control of Excess Light Energy in Photosystem II – The Phenomenon of Nonphotochemical Chlorophyll Fluorescence Quenching (NPQ) 214

9.4.2 The Molecular Components and Processes Involved in NPQ 217

9.4.3 Future of qE Research 238


10 What is in it for Plant, Biosphere and Mankind? 241 10.1 Science and Society 241

10.2 Energy Balance of Photosynthesis: A Wasteful Process? 242

10.3 Crops and Light Harvesting 247

10.4 Light Harvesting Principles for Future Applications: Liberation

from Saturation Constraints 249

10.5 Effects of Changing Climate – The Onset of Disorder 253

Bibliography 254

11 Conclusions 257

Index 261

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The science of biophysics of light harvesting has accumulated vast amount of knowledge

in the last 20 years about the workings of the photosynthetic membrane of higher plants.

The subject is a manifestation of a grand molecular design of the complex photosynthetic

machinery performing a sequence of primary energy transformation events. Hence, it

represents an excellent example for learning the principles of structure and mechanisms of

functioning of membrane proteins, their interactions with each other and their cofactors,

dynamics in the membrane and in the isolated state and various mechanisms of adaptation

to the environment. The author’s own 30 years’ experience in the field of biophysics of

photosynthesis and work done by his numerous colleagues has been presented in the

context of gradual explanation of complexity, historical development and multidisciplinary

character of the subject, The Photosynthetic Membrane . The need for such a text is long

overdue, since it does not cover the whole photosynthesis but focuses on its light phase

processes, concentrating on the light harvesting: a well-structured and regulated process

that ensures phenomenal flexibility of adaptations of plants to light that are essential for

their survival. The book starts from a general introduction to the essential features of life;

one of the most important is the energy requirement that is fulfilled almost solely by sunlight.

Further, the advantages and peculiar physicochemical features of the nanoscale level of the

photosynthetic membrane organization are described and the general makeup is presented

setting the scene and preparing the reader for the detailed up-to-date description of numer-

ous methods of investigation of the photosynthetic membrane structure and functions,

light harvesting principles, atomic structure of light harvesting antenna complexes, the

macrostructural organization and integration of antennae within photosystem complexes,

dynamic nature of light harvesting proteins studied by various spectroscopies and adaptations

of the light harvesting machinery in the intact membrane to light. This book also contains

a chapter considering the potential of the educational and practical applications of the

knowledge obtained in studies of the photosynthetic membrane organization and light

harvesting processes. The author contemplates the role of the fundamental knowledge in

general and explains possible ways it can be used in crop science, solar energy utilization

by mankind and in solving the problems associated with the global climate change.

The author addresses this book, first of all, to the final year undergraduate students of

various biology specializations. Therefore, he makes all effort to adapt it to different levels

of training in chemistry and physics. The level of complexity gradually increases towards

the end of the book making it relevant to postgraduate students and for a broader audience

of those involved in photosynthesis research as well as bioenergetic membranes in general.

With this book, the author hopes to awake an interest of a broad audience of students,

scientists and those who are attracted to the phenomenon of energy transformation

processes in living nature and welcomes any feedback.

Preface

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Welch Schauspiel! Aber ach! Ein Schauspiel nur! Wo fass ich dich, unendliche Natur?

Euch Brüste, wo? Ihr Quellen alles Lebens ,

An denen Himmel und Erde hängt , Dahin die welke Brust sich drängt - Ihr quellt, ihr tränkt, und schmacht ich so vergebens?

(Ah! what a view! Alas and but a view! Where shall I, endless Nature, seize on you?

Ye breasts, and where? Ye sources of all life

On which the heaven, the earth depends ,

Towards which the withered bosom tends —

Ye nourish, flow; yet vain my thirsty strife?) Faust: Der Tragödie erster Teil

by

Johann Wolfgang von Goethe

For undergraduate and postgraduate students and also those with an interest in the molecular engines of life.

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The author likes to thank several of his colleagues, particularly Drs Matthew Johnson and

Christopher Duffy for their engagement in many discussions on various topics of this book

that helped him to shape and improve his ideas and the original outline. He thanks

Dr Matthew Johnson for reading almost all chapters and Dr Christopher Duffy for reading

Chapters 4, 5, 10 and 11, giving him a number of useful comments. Dr Matthew Johnson

is also acknowledged for preparing Figure 2.4. The author thanks Dr Christopher Duffy for

preparing Figures 5.11 and 5.12. The author also thanks Professor Leonas Valkunas for

reading Chapters 5, 8 and 9 and giving him various useful comments. The author is grateful

to Professor Conrad Mullineaux and Dr Tomasz Goral for providing confocal microscopy

material for Figures 3.1(b), 3.2(b), a freeze-fracture electron microscopy image of

Figure 7.1 and the fluorescence recovery after photobleaching results presented on

Figure 9.17. The author thanks Dr Erica Belgio for preparing Figure 4.3 and Dr Erik

Murchie for providing him with the solar radiation spectrum for Figures 5.3, 10.2 and 10.4.

The author would like to thank Dr Rudi Berera, Dr John Kennis and Professor Rienk van

Grondelle for providing with a transient absorption spectrum of isolated LHCII trimer for

Figure 8.6. Dr Gene Carl Feldman is acknowledged for allowing him to use the NASA

satellite image of the global chlorophyll redistribution in the Biosphere. The author finally

thanks Mrs Kateryna Law for providing him with the image used for Figure 10.3.

The author appreciates the encouragement, help and advice received from the Wiley

publishing team; Paul Deards, Sarah Tilley and Rebecca Ralf.

Instructors can access PowerPoint files of the illustrations presented within this text, for

teaching, at: http://booksupport.wiley.com

Acknowledgements

Ruban_flast.indd xiii 8/31/2012 12:20:42 PM

1

The Photosynthetic Membrane: Molecular Mechanisms and Biophysics of Light Harvesting,

First Edition. Alexander Ruban.

© 2013 John Wiley & Sons, Ltd. Published 2013 by John Wiley & Sons, Ltd.

1.1 The Definition of Life

Since the Big Bang, the Universe went on a path of losing energy density, gaining entropy

and expanding, which causes continuing separation of its elements in space. Life seems to

be engaged in fighting these fundamental developments in the world ’ s fate by:

a. making the universal stuff more ordered , complex and predictable, hence defying the

laws of entropy, and

b. making the universe smaller by enabling the information flow and exchange constitut-

ing the multitude of reflections of matter via living creatures by their multiplication,

proliferation, memory of the makeup (genetic code), condensing matter; creation of

hypermolecules, as if exploring the potential and yet unknown properties of collectives

of the elements of matter.

In order to be successful in its apparently feeble refusal of matter ’ s fate, life is trying to

expand constantly in space and change in time. In spite of extreme environments, it

designs various ways to sustain this expansion process and invents new adaptations in

order to exist and continue to ‘harmonize’ the matter. In ordered and smaller space myri-

ads of choices can be realized, creating zillions of combinations of molecules, nonrepeat-

able events which are often perceived as increasingly deterministic as if there is a clever

plot behind it all. Instead, life is a chain of reflections copying matter, building more

complex matter and reproducing it. In order to do these unorthodox performances life

needs energy: a fundamental means to enable reflection, memorizing, building in its work

and multiplying.

Another feature of life is the ability to recycle biomolecules within the organism,

ecological niche and the entire Biosphere. Mankind generally does it, however, only in

Life, Energy and Light

1

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2 The Photosynthetic Membrane

extremely confined environments such as travel vessels: ships, submarines or spacecraft.

Life always uses the recycling principle.

Life is difficult to define in one or two simple sentences. Therefore, taking into consid-

eration all arguments provided previously we can present them in the ordered fashion in

form of a number of statements.

● Life is existence of protein and nucleic acid-containing autonomic open systems

exchanging energy and substance with environment in order to maintain their higher

levels of negative entropy (order) and proliferate in time and space. ● Life is largely based on molecular affinity, dialectics of attraction and repulsion forces

of the Universe, replicating itself using simple molecular coding principles, resulting in

constant evolution of dynamic molecular forms. ● In a way, life is a complex and far from equilibrium path towards condensing of matter. It

exists at rather low temperatures on the temperature scale achievable in the Universe.

Therefore whilst the high temperature irreversibly kills life the low temperature has a

tendency to preserve and even stimulate it in some cases (winter crops, Snowball Earth

theory, etc.). ● Currently, life seems to be impossible to create from the non-living substance, apparently

due to a long-term requirement for the evolution and selection of systems working

against the second law of thermodynamics. It is, however, likely that the selection process

which led to the creation of life forms is based upon a nonlinear chain of events and of a

fractal nature, that is can be characterized by sudden emergence of an infinite regularity

from some apparently chaotic and unpredictable trends. It cannot, therefore, be ignored

that tomorrow science would succeed in creating at least a primitive artificial life form. ● An essential prerequisite of life was the incidence of unlimited amount of easily

transferred in space energy (radiation), gradual cooling and condensing matter processes ● Life is a form of ‘revolt’ against the second law of thermodynamics achieved by very

unstable, ‘vulnerable’ (soft matter) systems, a fragile lip of matter towards order and

high organization on the way to the thermodynamic equilibrium and energy drain in the

expanding Universe. ● Life is an inherent property of our Universe and therefore is potentially as old as it is.

It is important to think of life origins from the thermodynamic point of view. In the end,

energy supply is a decisive factor for life in general and mankind, in particular.

1.2 The Energy of Matter

Energy equals matter and matter carries a lot of energy:

= 2E mc (1.1)

And this little formula means a lot for the Universe and life. Of universal energy, life

requires very little. If the energy hidden in a tiny nucleus is about 1 GJ per mole, the

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Life, Energy and Light 3

energy stored in the most common biological energy carrier, ATP, is 30.5 kJ per mole,

which is nearly 200 000 times smaller than the energy carried by tiny nucleus. Hence, the

question of energy supply to life should not be such a big deal. In a world of forces build-

ing the very blocks of the Universe; nuclei, atoms and molecules, the forces enabling life

are the weakest:

~1 GJ mol –1 ~1 MJ mol –1 500 kJ ------- 2–20 kJ - <1 kJ mol –1

Nuclear Atomic Molecular (covalent) Hydrogen – van der Waals

Forces of life

Fortunately for life, elemental and atomic energy transformations generate one of the by-

products – electromagnetic radiation – a broad range, speed-of-light travelling patches of

energy, capable to interact with matter. This is the best wireless and custom-addressed (not

all matter stuff can get it) form of energy ever known to man. Quanta of electromagnetic

radiation are spanning our Universe. Stars are the major sources of them. We sometimes

look at them at night, just registering small coloured sparkles of light – all energy is left for

us on the path of billions of years of its travel, just barely enough to cause a simple photo-

biological act of the retinal isomerization initiating the chain of the events of vision in our

eyes – all that remains for us from the mighty energy of a star.

1.2.1 The Source of Life ’ s Energy

What if the star can be brought to us a little bit closer, say a few light years or so? The one

we see every morning – the weather permitting – our Sun. People always wondered if they

have to thank it for something a bit more than just daylight, a suntan and warm weather.

Indeed, the Sun is giving to Earth 100 000 TJ of energy every second, a little more than that

required to tan our skins. It is actually enough to boil 100 thousands of billions (10 14 ) of

kettles, roughly 10 thousands per capita of the planet ’ s population. For someone preferring

a Bugatti-Veyron to tea, this is enough to run 20 Bugattis per person; mind you, it has got

to be a car driving on a ‘green’ fuel! But here comes the limited amount of space on our

planet to host not only all those cars but us, mankind with our tendency to scavenge the

nature which brought us into this world.

1.3 Energy for the Future

The point is that the Sun was, is and will be for some time, a very charitable body in the

sky: it gives us all this energy for free, unlike the energy supplying companies. The ques-

tion is how can we use just a tiny fraction of it in order to be alive and happy, driving our

modest vehicles, being curious about the word around us? Let us leave the question of our

lifestyle for a moment and think of how we can use the Sun ’ s generous energy? Naturally,

from solar cells: the devices capable of converting the light energy into electricity, a variety

of photovoltaic gadgets, which use the principles of photon energy conversion into the

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energy of moving electron or electric current. The industry is growing, in some countries

faster than in the others, and is certainly the way towards the era of recycling, renewal of

goods and energy for the future generations to come. Returning to life we may guess as

well how it has solved the question of energy supply in order to exist in the luxury of the

Biosphere: a carefully settled thin film of organic matter around the globe. Indeed, the key

to fixing all global crises is not just to find a quantity of matter or energy but to find the

ultimate way to live in balance in limited space and conditions. Indeed, the laws of

Biosphere do require the kind of lifestyle that is based not only upon the renewable energy

utilization but also uses a great biological principle of recycling substances. Without

acknowledging these laws one cannot succeed in sustaining life, including our species,

Homo sapiens . We have to find the solutions Nature found some 3 billion years ago. This

solution was building the living matter using the energy of Sun and later its most successful

variety; oxygenic photosynthesis .

1.4 Photosynthesis by Life

Photosynthesis is a process of conversion of energy of light into chemical energy of organic

compounds, carbohydrates. Oxygenic photosynthesis uses water as an electron donor for

redox reactions involved in the primary light energy stabilization.

+ + = +2 2 2 2( )CO H O hv CH O O

(1.2)

Photosynthesis is a process by which organisms capture and store energy of light by a

series of events that converts it into biochemical form of energy. Photosynthesis is a pro-

cess that is directed to increase levels of negative entropy (order) of living forms.

1( ) ,BS K ln

W− ⎛ ⎞= ⎜ ⎟⎝ ⎠

(1.3)

where (– S ) is negative entropy, k B is the Boltzmann constant and W is a number of possible

states the system can exist. W is also proportional to amount of information required to

describe the whole system and its dynamics. The more disorganized is the system, the less

predictable it becomes, and hence, it will require a lot of information in order to describe

all possible states the system can adopt and/or move into in time. Biological matter needs

input of negative entropy in form of energy and substance: hence, it must exist in an open

form. The energy type needs to be an ‘organized’ one, not just thermal or mechanic (lowest

types) but electromagnetic and/or chemical – ‘organized’ forms. The ‘organized’ character

of these forms comes from their specificity: they can be addressed to a specific atom,

molecule or group of molecules and cannot be ‘felt’ by the rest of the cell or organism.

Input of substances brings chemical form of energy as well as the material required for the

organism growth and reproduction.

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Life, Energy and Light 5

Photosynthesis has emerged on the very early stages of evolution. It remains one of the

most complex biological processes, which is not surprizing, since it holds the key to the

very heart of life: it is the means of fighting the universal entropy. How is the energy of a

tiny particle/wave, photon, used for this? The dualistic nature of something as casual as

light often appeared puzzling to scientists. A photon exhibits properties of both particle and

wave. Sometimes it is capable of propagating through two distant slits on the dark chamber

at the same time. Moreover, it has no rest mass. As Confucius said: ‘The hardest thing of

all is to find a black cat in a dark room, especially if there is no cat.’ Photosynthesis by

living organisms seems to be well-equipped to deal effectively with such an elusive form

of matter as light.

Photosynthesis is, first of all, a sequence of reactions of light energy transformation that

is evolved to solve two major tasks:

a. slowing down the reaction or rather the energy source, photon, and

b. stabilizing the captured energy, so that it cannot escape from the organism.

This looks something like catching a wild cat, making sure it gets calmer, slowing down

and is put safely into a space (cage, etc.) it cannot easily escape from. Catching a photon is

no lesser task for photosynthetic organisms. The principal difficulty here is to catch some-

thing, which has no mass when immobile, meaning that at rest it vanishes and ceases to

exist. How does life manage to slow the photon without killing it? Nature has found a neat

solution by using means of rapid transformation of photon energy.

1.4.1 Photon Energy Transformations

To catch the photon the pigment molecule ’ s optically-active electrons must react very

quickly, within 1 femtosecond (10 –15 s). Excited by light pigments can easily exchange their

energy of excited electron. This occurs via the electromagnetic resonance events, which

cause excitation energy wondering from one pigment to another, exciting without direct

molecular interaction. The energy is kept among the ‘collective’ of pigments and is waiting

for its fate, which can be various. The photon can reappear again, having less energy than

the absorbed one. Alternatively, the energy of a pigment can simply be wasted into heat,

contributing to the rise in the Universe ’ s entropy. Also, the electron spin can change to the

opposite creating so-called tripled excited state. Finally, the energy can be trapped by the

photochemically active pigment of the reaction centre to initiate the chain of electron and

coupled proton transport events leading to the chemical storage of light energy in the two

final products of the light phase of photosynthesis, ATP and NADPH (Figure 1.1 ). Those

can later be used elsewhere and when light is no longer present. These two substances are

the universal biological currency, always in demand for the ‘dark’ photosynthesis to fix

carbon from carbon dioxide of the air or just a synthesis of biological matter. Light harvest-

ing is therefore a staged process of photon-exciton-electron-proton transformations, han-

dling the most elementary and fundamental forms of energy and matter. Light harvesting is

the essence of photosynthesis in our planet. Remarkably, this energy transformations and

stabilization occur in one tiny kind of site, more specifically, surface, called the photosyn-thetic membrane .

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This book will talk about the composition, structure, variety of functions, adaptations,

assembly, biological importance and ways to study and to understand the photosynthetic membrane as the oldest and, so far, greatest light-harvesting nanostructure ever existed on

our planet that supported and continues to support all its life.

Reference

Confucius ( 1979 ) Analects . Penguin Books .

Bibliography

Barber , J. ( 2007 ) Biological Solar Energy . Phil. Trans. R. Soc. A , 365 , 1007 – 1023 . Blankenship , R. ( 2002 ) Molecular Mechanisms of Photosynthesis . London : Blackwell Science . Clayton , R.K. ( 1980 ) Photosynthesis: Physical Mechanisms and Chemical Patterns . Cambridge :

Cambridge University Press . Gleick , J. ( 1998 ) Chaos: Making a New Science . Vintage . Hall , D.O. and Rao , K.K. ( 1995 ) Photosynthesis . Cambridge : Cambridge University Press . Schrödinger , E. ( 1992 ) What is Life? With Mind and Matter and Autobiographical Sketches .

Cambridge : Cambridge University Press . Vernadsky , V.I. ( 1997 ) The Biosphere: Complete Annotated Edition . Heidelberg : Springer . Walker , D. ( 1992 ) Energy, Plant, Man . Brighton : Oxygraphics .

Light capture

Captured energy transfer tophotochemical converter–reaction centre

Electron transport

NADPH

>102 s

ATP

Proton transport

10–15 S

10–10–10–1 S 10–3–10–1 S

10–10 S

Photon Exciton Electron Proton

(~10 –36 kg) (10 –30 kg) (1.7 ×10 –27 kg)

Chemical storage

Carbonfixation

Light energy accumulation processes: time scales

Figure 1.1 Light energy accumulation processes: a sequence of particle transformation events (a simple scheme).

Rupan_c01.indd 6 8/29/2012 5:39:31 PM




Adde parvum parvo magnus acervus erit (Add a little to a little and there will be a great heap)

Ovid

2.1 The Cell Concept: Fundamental Nature of Life

All life forms apart from those very primitive ones, which are dependent upon other life

forms to provide them with homes where they live and proliferate (viruses, etc.), have a

very common building block, the cell. We are not a solid mass of matter (remember the

T-1000 terminator in ‘Terminator 2’ built of liquid metal? Or the planet-brain Solaris: both

apparently a homogenous mass that is alive?). Hence, life forms are represented by one,

several or many cells. The fact reminds us that we have evolved from a unicellular organism.

Cells are usually small, one tenth of a millimetre. Why do they have to be so tiny? The

answers are many. The first is actually, why not? These cells still contain millions of various

molecules organized in dozens of different compartments or organelles. Hence, in spite of

their small size the cell is much larger than the individual biomolecules within it and

therefore can be a complex, heterogeneous and multifunctional structure.

Indeed, there is a lot of space in the microworld. Richard Feynman ( 1960 ) named

his lecture given at an annual meeting of American Physical Society in 1959: There ’ s plenty of room at the bottom . What did he mean? Well, this physicist ’ s jargon refers to the

geometrical scales of matter: ‘bottom’ is the lower limit of special dimension and top, the

higher limits of special dimension. The lower limit is obviously infinitely small,

asymptotically approaching zero. But near it resides the world which is named with the

very frequently used term nowadays, nanoworld . The dimensions there are within nanometres

The Space of the Cell

2

Rupan_c02.indd 7 8/29/2012 5:39:13 PM


range (10 −9 m). The borders are arbitrary and gradually drifting up to the micrometer scale

(10 −6 m) approaching the millimetre border. A border that arbitrarily divides these worlds

of nano and micro from macro: the scale of existence in human dimensions. Basically it is

an invisible versus visible worlds division. Hence, from the lower limit of our macroworld

down the bottom of the nanoworld there are approximately six orders of magnitude of

dimensional space. That is one million! The space scale it covers can be compared to the

space scale between the size of a man and a country like the United Kingdom. There was,

and is a lot going on in the country. Imagine an endless list of institutions such as history,

geography, politics, arts, industry, agriculture, nature, science, religion, family, and so on.

An entire universe of human existence! In the same respect, the cell is an entire universe of

fundamental life on this planet. It is the basis for all Nature ’ s and our activities on it.

Feynman was seriously fascinated by the biological design:

A biological system can be exceedingly small. Many of the cells are very tiny, but they are very active; they manufacture various substances; they walk around; they wiggle; and they do all kinds of marvellous things – all on a very small scale .

But why have all life ’ s engines been evolved on the nanoscale rather than macroscale? Is

there any advantage or even absolute requirement for life processes to be organized on a

tiny invisible scale, as if we are not meant to see and learn what is going on? Hidden from

the eye cellular processes enabling life to take place where some laws of physics are very

different from those of a visible the macroworld. At the nanoscale the forces of surface

tension in the cell prevail over the forces of gravity. Hence, the capillary effects enable trees

to deliver kilograms and tons of water and nutrients upwards against gravity. For humans it

would require the use of powerful pumps: a macroworld solution to the problem.

Another peculiarity of the cell environment is the very high viscosity and high surface

area to volume ratio. These features enable cell robustness and at the same time extremely

high rates of substance exchange with the environment providing cells with power required

for fast growth and division. Another advantage of the nanoscale cell design is the very

high rates of thermal conductance. Indeed, excess thermal energy released as a result of a

multitude of biochemical processes can quickly be removed into the environment without

altering or causing any harm to delicate balance of cellular processes, the vast majority of

which are temperature sensitive.

An additional property of the nanoworld is that electrical charge concentration and

dynamics can be extremely high. Since electricity is utilized by the cell in a number of very

important functions, charge concentration and dynamics are important factors ensuring the

effectiveness and power of such processes as signal transduction, biological movements

and energy generation. And finally, arguably the most important property of the nanoworld

is that in some cases it provides the right space for otherwise entropic processes to become

pseudo-entropic or even organized. The fundamental grounds for this point can be seen

experimentally in the phenomenon of Brownian motion. There, the entropic motion of the

tiny pollen particles is driven by the collisions with a relatively small number of tiny

molecules of water, in the nanoworld the particle motion becomes visible simply because

Rupan_c02.indd 8 8/29/2012 5:39:14 PM

The Space of the Cell 9

of much smaller populations of water molecules are interacting with it. At a first glance,

Brownian motion is a problem for life. However, the entropic energy of spontaneous

molecular movements is utilized by life throughout, simply because it introduces certain

structural restrictions that can be imposed upon apparently stochastic processes. The

energy of molecular vibrations together with the energy of various gradients (that will be

described in the next chapter) is used to drive highly autonomic and directed processes in

the cell. Therefore these processes are subject to nonequilibrium thermodynamics, an

instrument to describe and understand the mechanics of molecular processes of life. Later

this point will become more apparent with the introduction of macromolecules and their

works in the nanoworld of the cell. The first chapter, describing the features of life, stated

that one of the major properties was the lowering levels of entropy in life forms. The

nanoscale seems to be a perfect field for this very fundamental feature of life in the

elementary space of the cell.

2.2 Compartmentalization: The Cult of the Membrane

When the space boundaries are defined like those of the ticket hole of the railway station,

where a crowd of people moves in unpredictable manner, the system looks highly entropic,

disorganized. How to make it more organized? The answer could be to create various spe-

cialized sub-spaces. Then the system would look more organized so that one should be able

to trace the reasons for people ’ s movements and behaviour in those different compartments

of space. The cell is a much compartmentalized structure. The walls of compartments are

made of similar structures to the wall of the cell – the lipid bilayer membrane – a remark-

able example of biological self assembly system. The order starts with establishment of

defined molecular interactions forming condensed states, like a plane made of a double

layer of molecules. Why a double layer? From inorganic chemistry we know that some

reactions lead to formation of water insoluble salts which form sediments of various shapes

or even organized crystals on the bottom of a test tube, a simple self-organizing system.

The property that made salts form those pellets is the stronger interaction between salt

molecules then between salt and water molecules. A simple but remarkable birth of order!

Like honey that attracts flies by making them all fly in one direction, abandoning their

chaotic movements in warm, summer afternoon light. One single property determines the

shape of substance in the water phase.

Molecules that form membranes, lipids, have a higher affinity to each other than to the

water molecules, therefore they form structures. But the 2D shape of the membrane and its

bilayer structure is determined by one additional property of the lipid molecule:

amphiphilicity or possession of both, hydrophobic and hydrophilic properties. Indeed, a

lipid molecule has one or more long hydrocarbon linear tails as well a hydrophilic head

built of oxygen-containing glycerol, alcohols, sugars or simply hydroxyl groups. In

addition, the head is attached to the tail via ester linkages containing ester and carbonyl

oxygen. Some lipids can contain dozens of oxygen atoms. Therefore, during lipid

interactions in the water phase, a bilayer structure is formed as a lowest energy

Rupan_c02.indd 9 8/29/2012 5:39:14 PM


thermodynamic state where hydrophobic fatty acid chains interact with each other via van

der Waals forces and are hidden from water and hydrophilic heads are exposed to water and

interact with it via hydrogen bonds. Hence, specific molecular properties and weak

interaction forces enable the process of biological self assembly based entirely on reaching

the thermodynamic energy minimum in the water system.

Membrane self-assembly process is driven by weakest molecular forces that define

hydrophilicity and hydrophobicity of lipid molecular structures. This is easy to see in the

simple computer stimulation experiment (Figure 2.1 ) that uses a molecular docking

algorithm. Here the van der Waals interactions are driven by the energy drop of a few

kcal mol -1 in the simple associate of two short fatty acid molecules (laurate) enabling

hydrophobic parts of the molecules to align for the maximum interactive configuration.

In the water phase the polar (hydrophilic) oxygen-containing groups (depicted in darker

shading) would in turn interact with water via much stronger then van der Waals hydro-

gen bonds. These interactions would further stabilize lipid associations (Figure 2.2 ) so

that they self-organize into bilayer structures reaching the stable energy minimum of the

whole system carefully dividing the structure and forces defining hydrophobic and

hydrophilic domains.

Because the biological membrane is built of the weakest molecular interaction forces it

is a very dynamic structure. Lipid molecules are free to move laterally relative to each

other, since at ambient temperature the entropy will provide enough energy to break (and

re-establish again and again) van der Waals interactions, making them change all the time

without compromising the overall stability of the membrane structure and keeping the

system at a relatively stable minimum energy. Increasing the ambient temperature will

inevitably lead to the increase in molecular movement within the membrane making it

more dynamic and will eventually exceed the membrane stabilization forces causing

damage and disassembly. Hence, the types and structure of molecules that build the

biological membrane could define its robustness and resilience to the environmental

temperature. One of the adaptive ‘measures’ could be to increase the area of van der Waals

surfaces. This can be achieved by increase in saturation of fatty acids making the ‘tail’

structures more flexible and ready to adopt the very minimum of collective lipid energy. In

addition, this surface could be enhanced by introduction of methyl groups into fatty acids.

Energy drop

2 kcal–1 Mol–1

Figure 2.1 The forces driving biological membrane self-assembly: calculation of energy drop during interaction of two molecules of laurate.

Rupan_c02.indd 10 8/29/2012 5:39:14 PM


It is achieved in membranes of Archaea . As a result these organisms can leave at extremely

high environmental temperatures, up to 80 °C and even higher.

The biological membrane is a typical and primary example of molecular antagonism that

leads to the automatic formation of biological structure. Perhaps the emergence of life in

the water environment was preceded by the formation of organic molecules that possessed

amphiphilic properties: amphibious characters, accommodating an inherent antagonism

between hydrophilia and hydrophobia. It will be further revealed, using the examples of

protein structures, that living nature is well designed for exploring thermodynamics in its

use to minimize levels of entropy and hence organize itself. The variety of lipid types offers

the biological membrane some flexibility in properties, such as fluidity, interactions with

environment, permeability, electric potential and accommodation of nonlipid membrane

components, such as sugars and proteins.

The biological membrane is a two-dimensional structure, but its space is very small. It is

a nanoscale structure that inevitably (thermodynamically predetermined) encapsulates a

closed space forming a vesicular structure, a prototype of the cell. The cell is an entire

fortress. Take, for example, The Tower of London. The cell and the Tower have a lot in

common. They have their entities marked and preserved by a barrier, wall or membrane.

The wall of the Tower defended it from invaders, enabled traffic through its gates, served

for observation of exterior environments. The cell membrane performs all of these functions

and in addition, in some cases even more. It can act as a kitchen, a place where new

substances are being synthesized and the biological energy can be produced. Within the

Tower stand a few buildings of various functions. Similarly, the fortress-cell has a variety

Hydrophilic phase

Integral protein

Lipids

Hydrophobic phase

Hydrophilic phase

Hydrophilicdomain

Membrane spanningdomain

Integralnonspanning

Peripheral

Figure 2.2 Protein stabilization in the biological membrane. Atoms in molecules are bound by covalent forces of about 100 kcal mol -1 . Hydrophilic domains of a membrane protein interact with the lipid head groups with electrostatic (10–40 kcal mol -1 ) and hydrogen (1–10 kcal mol −1 ) bonds. The hydrophobic fatty acid tails and protein domains interact via Dynamic van der Waals forces (0.1–1 kcal mol -1 ).

Rupan_c02.indd 11 8/29/2012 5:39:14 PM


of subcellular structures – organelles – surrounded by their own membranes. The cell is

packed with these structures so densely as houses in medieval town (take the Shambles of

York, for example). The crowdie compartmentalized nature of the cell interior is a crucial

feature of life design. Divide et impera or ‘divide and rule’: that was the key strategic

doctrine of the Roman Empire. In other words, segregation makes things better organized

and controlled, hence it allows a certain kind of order to be established.

The way cellular compartments are organized and their work is coordinated is remarkable.

The emperor is obviously the cell nucleus, sending various messages and directives to all

cellular compartments, it holds the information needed to build the cell structures.

Compartmentalization also takes place on the level of individual organelles and their

components. Membranes divide compartments and components and enable formation of

gradients of various substances: molecules, ions, protons, electrons and even electric fields.

The gradients accumulate potential energy, which when transformed into kinetic energy

can produce work and directionality, leading to formation of biological matter and

establishment of those functions characteristic for an organelle or entire cell. Order emerges

as a result. Barriers also serve to separate those types of molecules that otherwise would

interact with each other producing unwanted substances that may harm the cell and create

chaos. But what enables the membrane to possess such a variety of functions supporting

the life of the cell, tissue, organ and organism? The answer is membrane proteins and their

complexes with lipids and other functionally important cofactors, like ions and relatively

small organic molecules.

2.3 Membrane Components: Fundamentals of Proteins

The membrane is not much less complex and, by all means, not less fundamental than the

cell itself. In order to fulfil all those functions described in the previous paragraph it must

possess some specialized features. These features first of all, are represented by membrane-

associated proteins. Proteins in the membrane play a central role in enabling the biological

functions on a molecular level. Membrane proteins are a special class of proteins, which

are amongst the most difficult to study, mostly because the membrane is their natural

environment from which they cannot be harmlessly liberated without consequences for

their intactness. The membrane protein environment is amphiphilic (see Figure 2.2 ).

The presence of proteins in the membrane not only defines its functions but also

significantly changes its physicochemical properties, including temperature stability and

permeability to various molecules. Different biological membranes contain various

amounts of proteins. Myelin, a membrane that insulates nerve fibres has low protein content

(~18%). Relatively pure lipids are good insulators. In contrast, the plasma membranes of

most other cells are much more metabolically active and hence require more specialized

molecular apparatus. These membranes contain many pumps, channels, receptors and

enzymes: all proteins. The protein content of some plasma membranes is typically 50%.

However, biological energy-transduction membranes, such as the internal membranes of

mitochondria and indeed photosynthetic membranes have the highest content of protein,

Rupan_c02.indd 12 8/29/2012 5:39:15 PM


typically 75% and even more in some cases. Figure 2.3 displays electron microscopy

pictures of the fragments of photosynthetic membrane from higher plants containing two

types of proteins or rather protein complexes, photosystem II (PSII) and light harvesting

complex II (LHCII). It shows that proteins of different type and function can have different

density in the membrane. Whilst PSII particles positioned relatively far one from other

(more then a particle diameter), the LHCII particles look like a crowd of proteins, tightly

interacting with each other. This simple observation could be indicative of the different

functional roles of the two types of membrane proteins.

Whilst membranes of specialized cells often contain only a few types of proteins,

membranes of metabolically active cells and organelles normally contain a large variety of

protein types, structures and sizes. These proteins can also vary in the way they interact

with or are positioned within the membrane. Typically membrane proteins span the whole

width of the membrane having a hydrophobic membrane-spanning (transmembrane)

domain and two hydrophilic domains protruding into water phase (see Figure 2.2 ). These

proteins are called integral membrane-spanning proteins. There are, however, proteins, that

are integral but do not span the whole membrane depth. They are called integral non-spanning membrane proteins. The other type of proteins does not even interact with the

hydrophobic lipid interior of the membrane. They are simply bound to the hydrophilic

domain of other membrane proteins via most commonly ionic interactive forces and

hydrogen bonds. This type of interaction is relatively strong and normally these proteins

are fixed permanently in their binding site and do not move around, whilst integral

membrane proteins display a certain degree of mobility (this will be discussed in detail

later). The ability of membrane proteins to move in the membrane plain allows them to

interact with each other, regroup, oligomerize and form domains, thus giving the membrane

a heterogeneous character. All this enables a broad range of adaptations of the membrane

structure and function suited to various metabolic and environmental changes and

requirements.

Figure 2.3 Different densities of two types of membrane proteins. Freeze-fracture electron microscopy image of the photosystem II (left) and LHCII particles.

Rupan_c02.indd 13 8/29/2012 5:39:15 PM


One important feature of membrane proteins is that they very often form relatively

stable superstructures: complexes of several subunits (sometimes dozens of subunits). The

molecular weight of these complexes can reach hundreds of thousands and even a few

millions of Daltons. Those who work on the evolutionary aspects of membrane protein

functions are often puzzled by the question of how at all such complex, cooperatively built

and functioning molecular machinery could evolve? What is even more intriguing is that

whilst some of these structures took a relatively short time to evolve, they have been then

preserved in nature for hundreds of millions of years.

Another interesting aspect of the superstructural character of many membrane proteins

is that since they are very large (sometimes 10 or even 30 nm in diameter) there are not so

many of them in the biological membranes of organelles, in particular, to treat their

behaviour (movements and interactions) with random statistics, there are simply too few of

them and they do not behave any more as billions of small molecules obeying the laws of

classical molecular thermodynamics. There only are about 200 PSII complexes in the area

of 400 × 700 nm of the photosynthetic membrane presented in Figure 2.4 . Hence, it is not

surprising to see otherwise improbable, nearly perfect transient ordering of membrane

supercomplexes as displayed here. The behaviour of such system cannot be correctly

described by statistical molecular physics. It would require N-body modelling approaches

used in the studies of dynamics of stellar clusters in the Universe, a totally unexpected turn

in the research of a nanoworld of the biological membrane. Indeed, for such a large structure

as the PSII complex factors like symmetry (or indeed asymmetry), sensitivity to

environment, ability to interact via various types of weak forces (see the Figure 2.2 caption)

could be major determinants of 2D diffusion and, hence, positioning. Therefore, the

conventional statistics of molecular behaviour are replaced in the membrane by a more

deterministic control resulting in predictable collective dynamics and simple control over

function, and in this way establishing order in the nanoworld of cell processes, an ultimate

goal of life.

Figure 2.4 Order versus disorder of membrane protein assemblies. Freeze-fracture electron microscopy image of the photosystem II-containing membrane. Courtesy of Matt Johnson.

Rupan_c02.indd 14 8/29/2012 5:39:15 PM


2.4 Functional Classification of Membrane Proteins

Proteins in the membrane play a number of vital cellular and organellar functions. These

functions can be divided into the three major groups: transport , signalling (reception) and

metabolism (enzymes).

Transport proteins are the largest and the most versatile in functions and structure group

(Figure 2.5 ). It consists of photosynthetic light harvesting antenna complexes , which ‘deal’

with the tiniest particle, the ‘photon’ that carries the energy needed to drive the photosyn-

thetic reactions. The second group of transport proteins is light driven proton pumps . The

typical example of this group is bacteriorhodopsin, a protein that captures light energy and

converts it into the energy of a proton gradient across the membrane it is located in. Hence,

this is a complex transporter that deals not only with photons but also much larger particles,

protons. The next group of transporters are light-driven electron transport complexes .

These are represented by reaction centre complexes of various photosystems of prokary-

otes to eukaryotes. The role of these complexes of proteins is to convert the energy of

excited by light electrons into the energy of moving electrons that are donated into the

chain of reactions of reduction and oxidation that supply the cell with NADPH and ATP.

Another group of transporters is electrochemical potential transporters . Typical represent-

atives are proton-drive ATPases that transform energy of the proton gradient across the

1 Light harvesting complexes

Antennae of all photosysthetic organisms

Bacteriorhodopsin-like proteins

Photosynthetic reaction centres

Cytochrome b6f complex

Proton-driven ATPases

Calcium ATPase

Voltage-gated ion channels

Drug/Metabolite transporters

GPCRs (Rhodopsin), GABA receptors (gamma-aminobutyric acid)

Prostaglandin H synthase, Protoporphyrinogen IX oxidase

2 Light absorption-driven pumps

3 Light-driven electron transfer complexes

4 Electron transfer complexes

5 Electrochemical potential transporters

6 Phosphate bond hydrolysis-driven transporters

7 Ion channels

8 Porters (uniporters, symporters, antiporters) and porins

9 Receptor proteins

10 Enzymes

TR

AN

SP

OR

TE

RS

LHCII Photosystem II PRIMARY ENERGY

PRIMARY & METABOLICENERGY

WORK

COMMUNICATIONS

METABOLISM

SIGNALLING

METABOLISM

Bacteriorhodopsin

Cytochrome b6f H+-ATPase

PHOTOSYNTHESIS

PHOTOSYNTHESIS& RESPIRATION

Figure 2.5 Functional classification of membrane proteins.

Rupan_c02.indd 15 8/29/2012 5:39:15 PM


membrane they are inserted in to synthesize ATP from ADP. Relative to them is a group of

transporters that work in reverse, hydrolysing ATP in order to transport various ions across

the membrane (calcium ATPase). The other group of transporters is essential for channel-

ling ions ( ion channels ). Next group of transporters is capable of transporting molecules of

various sizes actively ( porters ) or passively ( porins ).

Functions of transport membrane proteins span from primary to metabolic energy gen-

eration, from work, communications to cellular homeostasis. A remarkable feature of

transport proteins is that they are capable of handling the particles of matter that vary in

sizes by more than 10 orders of magnitude (mass of a moving photon is ~ 10 −33 g, mass of

electron is ~ 10 −27 whilst the mass of glucose is ~ 3 . 10 −22 g). This is yet another feature and

advantage of a nanoscale design of life.

The second class of membrane proteins are various receptors that form systems of

cellular communication and signalling (G-protein coupled receptors (GPCRs), gamma-

aminobutyric acid (GABA) receptors). Here, we see a very similar feature to transporter

proteins: ability of receptor proteins to ‘handle’ particles of nature from photon of light and

ions to large organic molecules and even proteins.

The third class of membrane proteins are enzymes . A typical example is prostagandin H2

synthase that catalyses the first step in prostaglandin synthesis. The enzyme is an integral

but nonspanning membrane protein. Its active centre must be located within the hydropho-

bic environment, since the substrate is a fatty acid (arachidonate). The hydrophobicity of

the substrate simply dictates the design and location of the metabolic enzyme, not in the

water phase but in the 2D space of the membrane interior.

Reference

Feynman , R. ( 1960 ) There ’ s Plenty of Room at the Bottom . Caltech Engineering and Science , 23 , 22 – 36 .

Bibliography

Berg , J.M. , Tymoczko , J.L. and Stryer , L. ( 2012 ) Biochemistry . Basingstoke : W.H. Freeman and Company .

Branden , C. and Tooze , J. ( 1991 ) Introduction to Protein Structure . New York and London : Garland Publishing .

Luckey , M. ( 2008 ) Membrane Structural Biology (With Biochemical and Biophysical Foundations) . Cambridge : Cambridge University Press .

Nicholls , D. and Ferguson , S.J. ( 2002 ) Bioenergetics . London : Academic Press .

Rupan_c02.indd 16 8/29/2012 5:39:15 PM




‘ In thy house or my house is half the world ’ s hoard …’ Rudyard Kipling

3.1 Knowledge of the Pre-Atomic Structure Era: Organization of the Photosynthetic Membrane System

Let us consider the photosynthetic membrane of the higher plants. Figure 3.1 (a) shows typical

evenly green young spinach plants. Spinach has been a model plant for research in photosyn-

thesis spanning from studies of primary processes to experiments on carbon assimilation in

isolated chloroplasts. They can be visualized even without preparation in the intact cell using

the modern confocal scanning fluorescence microscopy (see the next chapter for details).

Figure 3.1 (b) shows a confocal fluorescence microscopy image of round spinach chloroplasts,

a very high resolution optical microscopy, whilst Figure 3.1 (c) displays an image of isolated

chloroplasts obtained by conventional optical microscopy. It is easy to detect the intact chlo-

roplasts, which show much better contrast to the broken organelles, which loose their shape

and appear rather flat. Both images, however, reveal the presence of grained structures with

higher intensity of absorption/fluorescence inside of the chloroplast. They can be numerous

and are called granae. The medium they are embedded into is called stroma. The granum is a

The Photosynthetic Membrane: Outlook

3

Rupan_c03.indd 17 8/29/2012 5:38:57 PM


(a) (b)

(c) (d)

(e) (f)

(g) (h)

Chloroplast

Granae

Granae

Granal thylakoid

PSII

LHCII

PSII

Rupan_c03.indd 18 8/29/2012 5:38:58 PM

The Photosynthetic Membrane: Outlook 19

very structured particle that can be explored in detail only by a higher resolution microscopy,

like transmission electron microscopy (EM). EM images in Figure 3.1 (d) and (f) display

numerous grana in the chloroplast and a single granum that exhibits a clear lamellar structure.

Figure 3.1 (e) shows a schematic simplified presentation of the structure of granum. It is basi-

cally a stack of flattened often round vesicles called thylakoids. Vesicles are surrounded by

membranes, which, in fact, are the photosynthetic membranes of higher plants. The volume

insight the thylakoid is called the lumen. Its space is very narrow, only about 10 nm wide. The

physiological reasons for the existence of the lumen space, that is, a closed volume surrounded

by the photosynthetic membrane, will be explained later.

Stacking of thylakoids into granae is known to depend upon the presence of divalent

cations of magnesium, Mg 2+ . Monovalent cations of potassium, K + , are also effective

though at higher concentrations than Mg 2+ (100 mM of K + vs 5 mM for Mg 2+ ). It is generally

accepted that positively charged cations bind to negatively charged groups exposed on the

membrane surface therefore compensating electrostatically the screening repulsive forces

between membranes assisting thylakoid stacking. As will be shown later, membrane

proteins play crucial role in this process undergoing a radical lateral cooperative regrouping

in the thylakoid membrane system. Still, there are also thylakoids in chloroplasts that are

not involved in formation of granae called intergranal or unstacked thylakoids. Together

with granae they form the chloroplast thylakoids, a site where the whole light phase of

photosynthesis takes place. Thylakoids can be isolated relatively easy from chloroplasts

and have been the subject of studies for a number of decades. Some of them will be

mentioned later in this book. However, first of all it must be said that the thylakoid system

is the site in chloroplasts that plays a direct part in the absorption of sunlight and, hence,

the trapping photons. What is interesting is that the area of these photosynthetic membranes

is extremely large. Let us do a simple rough calculation.

Take 1 cm 2 of the average spinach leaf. Its area in micrometres would be 10 8 μm 2 . The

average cell size is about 20 × 40 μm, therefore the area is 800 μm 2 . Hence, one cell layer of

the 1 cm 2 of leaf will contain 125 000 cells. Each cell can carry ~ 50 chloroplasts (in

shade-grown plants). Each chloroplast can easily contain 100 granae with around seven

stacked thylakoids, each having two membrane surfaces. The average thylakoid diameter is

0.5 μm, which corresponds to ~0.2 μm 2 . Using these numbers we can calculate the total area

Figure 3.1 Origin and organization of the photosynthetic membrane of higher plants. (a) Spinach plants – a common model for mechanistic photosynthesis research. (b) Fluorescence confocal image of chloroplasts in the intact leaf showing clearly separated fluorescing dots – granae. Bar is 5 μm (courtesy of Tomasz Goral and Conrad Mullineaux). (c) Isolated chloroplasts in the incubation medium. Bar is 10 μm. (d) Electron micrograph of a single chloroplast showing dark elongated granae and large white starch granulae (centre). Bar is 1 mm. (e) Schematic presentation of thylakoid system structure of the chloroplast. (f) Electron micrograph of a single grana stack (centre). Bar is 200 nm. (g) Freeze-fracture electron micrograph of a single thylakoid membrane from grana showing photosystem II particles. Bar is 100 nm. (h) Freeze-fracture electron micrograph of grana thylakoid membranes displaying photosystem II as well as light harvesting complex particles (LHCII). Bar is 200 nm. (See Plate 3.1 in colour plate section.)

Rupan_c03.indd 19 8/29/2012 5:38:59 PM


of only granae membranes in the one layer of cells of 1 cm 2 leaf area. It yields approx.

20 cm 2 , more than an order of magnitude higher than the leaf area itself. Remarkably, this

is achieved despite the total chloroplast volume is being only ~1/10 of the cell volume. Taking

into account that the average leaf can contain around 20 cell layers we can see that the total

grana membrane of the leaf of 1 cm 2 can be about 400 cm 2 , that is, 20 × 20 cm. For a leaf with

10 cm 2 area, this will be 4000 cm 2 or nearly a half of a square metre! This is a truly remarkable

achievement of a compact character of the nanoscale organization of the chloroplast cell

compartment reflecting Feynman’s famous remark about the significant amount of space the

nanoworld potentially possesses (see the previous chapter). By making light harvesting take

place in a very thin two-dimensional system, the membrane, it is possible to compact a large

membrane area into a relatively small three-dimensional space. This space in the case of the

photosynthetic membrane area is well-utilized in the processes of adaptation to the light

environment that involve alterations in leaf, cell and chloroplast morphology. This is a well-

documented fact that plants acclimated to low light accumulate more chloroplasts per cell,

grana per chloroplasts and thylakoids per grana. This type of acclimation results in a one or

even two orders of magnitude increase in the light interception and photosynthetic capacity

of the plant, overall. The changes in the ‘macroworld’ of the plant also take place during light

acclimation manifesting in alterations in the leaf area, and so on. However, those occur on

much smaller scale than the changes in the structure on the level of organelles.

Another interesting feature of the efficiency in light interception by the leaf cell is that

chloroplasts can move and adopt positions to maximize or indeed minimize light

interception. The elements of structure such as cell walls and vacuoles scatter light

effectively in the leaf so that it can propagate virtually in any direction reaching optimal

numbers of chloroplasts and their thylakoids. Figure 3.2 , left , displays even redistribution

of chloroplasts in the spinach cell grown under low light environment. Figure 3.2 , right , shows the effectiveness of oak leaves absorption of sunlight. The second layer of leaves

Figure 3.2 Scattering and absorption of light by oak leaves. Left : 1, 2 and 3 order of leaves facing light. Right : chlorophyll fluorescence confocal image of the leaf cell showing even redistribution of chloroplasts. Bar is 5 micrometers (Photo on the right is courtesy of Tomasz Goral and Conrad Mullineaux). (See Plate 3.2 in colour plate section.)

Rupan_c03.indd 20 8/29/2012 5:38:59 PM


shaded by the first layer still receives some light, whilst the third remains in very shaded

environment. As it will be shown in a later chapter, filtration of light by leaves in diverse

and stratified ecosystems alters significantly spectral quality of light available for plants

that live under canopy of others and therefore demands and, indeed, results in development

of important light adaptation strategies by the photosynthetic membrane.

3.2 Composition of the Photosynthetic Membrane

3.2.1 Lipids

Lipids are essential components in the photosynthetic membrane. First, their amphipathic

properties direct the self assembly process leading to the formation of biological membranes

(see Chapter 2). Thylakoid lipids make up to 50% of a total membrane mass. Also, this

figure depends on the plant growth conditions and the stage of chloroplast development

and can often be rather smaller. Second, they are essential components of the membrane

protein complexes, helping to shape protein structure and function (see the next section).

There are four major classes of lipids present in the thylakoid membrane of plants. These

include two classes of galactolipids, mono- and digalactosyldiacylglycerides (MGDG and

DGDG, respectively). They are the major lipids in the thylakiod membrane, occurring at

the ratio of MGDG to DGDG ~2 : 1 and constituting up to 75% of the total lipid content

in spinach membranes. The remaining lipids of the thylakoid membrane are made up

of phosphatidylglycerol and sulfolipids, occurring in approx. 2 : 1 ratio. Structure of all

thylakoid membrane lipids is based upon glycerol, esterified by two fatty acids to carbons

1 and 2 (Figure 3.3 ). The most common fatty acid is linolenic, 18 : 3, which is nonsaturated

CH2OH

CH2OH

CH2SO3

HO

H

HOH

OH

OHHOH

H

HO

H

HOH

OH

OH

H

H O

O

O

OP O

OH

OHO

HO

H

OH

HOH

OH

OH

H

H

HO

DGDG

MGDG

SQDG

PG

O

O

Figure 3.3 Structure of the photosynthetic membrane lipids. MGDG, monogalactosil-diacylglyserol; DGDG, digalactosildiacylglycerol, PG, phosphatidylglycerol; SQDG, sulfoquinovosyldiacylglycerol.

Rupan_c03.indd 21 8/29/2012 5:39:00 PM


and can represent up to 95% of all types of fatty acids in particularly in galactolipoids of

spinach thyalakoid membranes. In addition, a bit shorter, hexadecatrienoic (16 : 3) and

hexadecatetraenoic (16 : 4) acids were found along with less saturated linolic (18 : 2) and

saturated, palmitic (16 : 0) acids can also be found in spinach thylakoid membranes. High

levels of nonsaturated fatty acid residues enhance greatly membrane fluidity. Regulation in

the ratio between saturated and nonsaturated fatty acids is a fundamental mechanism that

alters membrane fluidity and sensitivity of membrane functions to the environmental

temperature. In addition, the sheer diversity of fatty acid composition has been noticed

among various groups of photosynthetic organisms. The hydrogen atom of the third

glycerol carbon is replaced by the galactose (in MGDG), di-galactose (in DGDG),

sulfoquinovose (in SQDG) or glycerophosphate (in PG) (see Figure 3.3 ). There is a clear

asymmetry in redistribution of these four classes of lipids in the thylakoid membrane.

Galactolipids prefer to face the outer, stromal side of the membrane (up to 60% of

MGDG + DGDG). Phospho- and sulfolipids are mainly localized in the other half of the

bilayer with hydrophilic head groups facing lumen. It is likely that such asymmetry as well

as interactions with protein complexes hold the keys to the diversity and functions of the

photosynthetic membrane lipids.

3.2.2 Lipid-Related Compounds of the Photosynthetic Membrane

There exist a number of other lipid-like molecules in the thylakoid membrane of plants

(Figure 3.4 ). Most common and functionally sound are chlorophylls, the green pigments of

photosynthesis. Their concentration in the membrane is only approximately 2.5 times lower

then that of all lipids. Chlorophylls possess hydrophobic mostly saturated hydrocarbon tail,

phytol. Similarly to the hydrophobic tails of lipids from Archaea (see Chapter 2) the phytol

tail is methylated: this feature enhances the strength of hydrophobic interaction between

chlorophyll and other membrane components and therefore enhances structurally their

stability. The latter fact is important, as will be described later for the essential functions of

this pigment. The hydrophilic component of the chlorophyll molecule is the large macrocycle, chlorine ring. The functional reasons for this will be detailed later. As far as the

structure of the chlorine ring is concerned, it contains five oxygen atoms that mostly deter-

mine its polar nature and interactivity with the membrane components, mostly via hydrogen

bonds which are much stronger then van der Waals forces (see Chapter 1). Another key

structural feature of chlorophyll is the presence of magnesium atom in the centre of chlorine

ring coordinated by nitrogens of the pyrrole rings. Magnesium coordination number in vivo ,

as will be shown later, is normally five or even six due to additional interactions formed by

the atom and the groups in the environment. Since the coordination interactions are even

stronger than hydrogen bonds, magnesium plays a dominating role in determining

interactions of chlorophylls within the photosynthetic membrane.

The next class of lipid-like compounds is tocopherols. Their concentration is much

smaller than that of chlorophylls. The hydrophobic component is similar to saturated

methylated structures of phytol or fatty acid residues of Archaea . The hydrophobic part is

formed by a derivative of chromene – a heterocycle of benzene and pyrane rings. The

Rupan_c03.indd 22 8/29/2012 5:39:00 PM


hydroxyl group on benzene cycle is a functional group that can interact with free radicals

neutralizing them. Hence, tocopherols are essential membrane-associated radical

scavengers. The production of radical compounds is inevitable, particularly under various

stress conditions, in the photosynthetic membrane due to the numerous processes of redox

chemistry that are crucial for the light phase of photosynthesis (see the next paragraph).

There are several types of tocopherols ( α , β , γ and δ ). They differ by the types of residues

(methyl group or hydrogen) attached to the benzene part of chromanol.

Another functionally important class of lipid-like compounds is represented by quinones.

Their hydrophobic component is highly nonsaturated hydrocarbon structure made of

numerous isoprenoid residues. Basically, it can be seen as phytol or tocopherol tails that

acquired periodic double bonds. The presence of nonsaturated bonds within the hydrocarbon

tail of lipids normally leads to disruption in their interactions with environment and makes

Fatty acids

Chloroplylls

Tocopherols

Quinones

Xanthophylls

Detergents

C.O C16 16:1

Phytol

Plastoquinone

α-tocopherol

O O

O

O

O

HHH

H

H

O

HO

O

OH3C

CH3

H3CO

Lutein

b-D-glucoside n-octyl -β-D-glucoside

9

H

13 5

7 9 11 13 15

15′ 13′ 11′ 9′ 7′1′

5′ 3′OH

HO

O

O

b-D-glucosideRhodopin glucoside

Figure 3.4 Comparison of the structures of various lipid-related molecules.

Rupan_c03.indd 23 8/29/2012 5:39:00 PM


the structure more rigid, since the rotation around a double bond is very restricted in

comparison to that of a single C—C bond. This feature of a hydrophobic quinine tail

could be a key in enabling high rates of diffusion of this molecule, the essential feature of

plastoquinone function, since it works as a shuttle carrying electrons and protons between

different electron transport components of the thylakoid membrane (see the next paragraph).

The hydrophilic part is represented by the twice methylated p-benzoquinone derivative

(Figure 3.4 ). Benzoquinone carbonyls are functional groups involved in redox reactions of

electron transport of relatively low potential that receive two electrons and two protons and

carrying them to the electron acceptor. The concentration of plastoquinones is relatively

low, only 5–10% of the total lipid content.

Finally, oxygenated carotenoids, xanthophylls, form a group of accessory to

chlorophylls pigments in the photosynthetic membrane. In higher plant membranes

they are largely symmetric, containing two polar compounds attached to opposite ends

of the hydrophobic hydrocarbon structure derivative of polyenes that are methylated in

a similar fashion to phytol, tocopherols and plastoquinones defining their terpenoid

nature. However, the key difference of this structure is that as in all polyenes they con-

tain conjugated double bonds, unlike plastoquinones. The importance of conjugation in

xanthophylls and indeed chlorophylls will be explained in detail in a later chapter. The

structure of higher plant xanthophylls is rigid and can undergo various types of trans-cis isomerization. The polar end groups of xanthophylls are various derivatives of

cyclohexene that contains hydroxyl or epoxy groups. The symmetric nature of xantho-

phylls is often central in the orientation of these molecules within the membrane. They

can span the whole membrane depth with their polar groups oriented towards hydro-

philic residues of membrane components.

Variety of xanthophylls in photosynthetic organisms of different classes is impressive.

In lower organisms as photosynthetic purple bacteria xanthophyll structure can be very

similar to the structure of mild detergents glucosides. Figure 3.4 shows structures of

n-octyl- β - D-glucoside and carotenoid from Rodopseudomonas sphaeroides , rhodopin glu-

coside. Remarkably their polar groups are represented by the same structure, β -D-glucoside

residue. Both structures are asymmetric. The ‘tails’ are different, though. Whilst the

detergent possesses short saturated hydrocarbon chain (C8), the xanthophyll contains long

conjugated structure, typical for carotenoids. This structure is more rigid than that of the

detergent or indeed saturated fatty acids. Hence, Figure 3.4 displays various types of

amphiphilic molecules that reside in the photosynthetic membrane. Combination of both,

hydrophobic and hydrophilic properties ensures their affinity and orientation within the

lipid bilayer, whilst the hydrophilic groups carry important functions. Figure 3.5 shows an

example of interactions between three key types of described photosynthetic membrane

compounds, lipid, chlorophyll and xanthophyll. All three molecules interact mainly via

van der Waals forces, bringing their hydrocarbon tails in close almost orbital contact.

Whilst the carotenoid structure is in all- trans configuration rigidly spanning the hydropho-

bic environment, chlorophyll and in particular lipid residues are more freely bend embrac-

ing each other in less linear fashion. As it will be seen later, all three are accommodated by

a most crucial component of the photosynthetic membrane, a membrane protein.

Rupan_c03.indd 24 8/29/2012 5:39:00 PM


3.2.3 Proteins and Protein Complexes

Proteins are by far one of the most versatile and dominating group of molecules in the pho-

tosynthetic membrane. Certain membranes developed under particular conditions can be

built of up to 80% or even more of protein. According to the membrane protein classification

displayed in Figure 2.5 they all belong to the group of transporters. The process of photo-

synthesis starts with the antenna that is represented by a family of light harvesting com-

plexes; proteins that bind and organize pigments, chlorophylls and carotenoids as well as

lipids. These complexes absorb photons of light, the tiniest particle in nature; convert its

energy into the energy of excited electrons that can be transported via exciton energy trans-

fer into the second group of photosynthetic membrane complexes, reaction centres. The

latter donate electrons ‘charged’ with the energy of light into the chain of redox reactions

that transport electrons and protons along and across the membrane, correspondingly.

Proton gradient energy can be used by ATPase, another transport-enzyme complex that

synthesizes ATP from ADP and phosphate. Figure 3.6 shows a sequence of particle trans-

port events that takes place in the thylakoid membrane of plants. The sheer range of particle

masses that these proteins handle can be understood by comparing the sizes in the macrow-

orld. Assuming that the photon corresponds to the size of an ant, electron would correspond

to the size of a rabbit, whilst proton would be as large as a car. The average size of a mem-

brane protein would be as big as the largest excavator (Bagger 288) or a train that weighs

more than 10 thousand tons. Hence, the membrane proteins must be built with such a preci-

sion that they can handle accurately much smaller, albeit, highly energized forms of matter,

transporting them between each other and converting its energy into the chemical energy of

primary photosynthetic products, NADPH and ATP. It is amazing that there is enough room

in the biological membrane nanoworld to accommodate structures that handle particles

with high precision that are smaller than themselves by 13 orders of magnitude!

Chlorophyll

Lipid

Xanthophyll

Figure 3.5 An example of close van der Waals interactions between a fatty acid residue of phospholipid, chlorophyll phytol tail and xanthophyll. The coordinates were taken from the structure of the LHCII complex (for details see Chapter 6). (See Plate 3.5 in colour plate section.)

Rupan_c03.indd 25 8/29/2012 5:39:00 PM


Figure 3.7 displays a scheme of organization and functional interactions between

the thylakoid membrane complexes. There are basically five major photosynthetic

complexes:

1. Light harvesting antennae;

2. Photosystem II (PSII);

3. Cytochrome b6/f complex;

4. Photosystem I (PSI);

5. ATPase.

In the early days of photosynthesis research it was believed that chlorophyll was freely

located in the membrane among lipids. However, later it was shown that all chlorophyll is

attached to three major classes of membrane protein; light harvesting antenna, PSI and

PSII. Later, each photosystem was found to possess its own antenna in a form of a several

related pigment-protein complexes. Photosystem I contains at least four types of LHCI

antenna polypeptides, called Lhca1-4. Photosystem II has at least six types; Lhcb1-6.

These polypeptides form light harvesting antenna or outer antenna. They assemble into the

major, trimeric complex, LHCIIb (Lhcb1-3) and three minor monomeric complexes

LHCIIa (CP29), LHCIc (CP26) and LHCIId (CP24) (for more details see Chapters 6 and 7).

They are organized around core complexes of photosystems. These complexes are built

with a large number of subunits. The PSII core complex contains at least 23 polypeptides,

whilst complete with LHCI PSI complex contains 17 polypeptides. The subunit sizes of

photosystems vary from a few kD to 50-70 kD. Core complexes contain inner antenna

polypeptides, CP43 (PsbC) and CP47 (PsbB) in PSII and PsaA and PsaB in PSI. They carry

only one type of chlorophyll, chlorophyll a , whilst LHC complexes carry also chlorophyll b .

Once light energy is delivered to PSI and PSII by means of a resonance energy transfer, it

is passed to the reaction centre chlorophyll via their inner antenna. The inner antenna chlo-

rophyll is supposed to focus the excitation energy onto the reaction centre. The reaction

centre of photosystem II (P680) is organized as a chlorophyll dimer, the spatial pair, located

Photon

~10–33 g 1·10–27g 1.7·10–24g

Exciton Electron Proton

Light harvestingantenna

Electron transferproteins

ATPase

1 mgant

1 kgrabbit

Membrane protein: >10 Kton(train or Excavator 288)

1.7 toncar

Figure 3.6 Classes of photosynthetic transporter proteins handling various particles of matter.

Rupan_c03.indd 26 8/29/2012 5:39:01 PM


on D1 (PsbA) and D2 (PsbB) polypeptides. Upon excitation RCII is capable of donating an

electron into the electron transfer chain first to pheophytin, then to bound plastoquinone

(QA-site on D2 protein) and finally dissociable plastoquinone (QB-site on D1 protein). For

dissociation from the QB-site it requires two electrons and two protons to bind to

plastoquinone that diffuses in the membrane to be bound to the cytochrome b6/f complex.

After electron donation, P680 + is acting as a very strong oxidant (oxidation potential ~1.17 V),

capable to remove an electron from water, via the oxygen evolving complex (OEC) that is

represented by a group of three extrinsic membrane proteins, PsbO-Q. The electron transfer

in PSII is linked to a proton discharge into the thylakoid lumen in a sequence of water-

splitting reactions in OEC.

The other site of the proton release into the lumen is cytochrome b 6/ f complex. This

complex is built at least of eight proteins. Four of them play defined functions. When

doubly reduced plastoquinone binds to this complex, near to the lumen (Q-site), it gives

Terminal thylakoids

MarginStroma

NADP

2H+

2H+2H+ FNR

Fde–

e–

e–

e–

2e–

2e–

PQPQ

RC

PC

FeSRC

OEC

H2O 4H+ 2H+O2

PQH2

PQ

NADPHADP+Pi

ATP

PSII

Lumen

Lumen

LHCIIb LHCIIba,c,d

PSII Cytb6/f PSI LHCI ATPase

Figure 3.7 Complexes of the photosynthetic membrane. Energy transformation chain of the light phase of photosynthesis: photophysical, photochemical, redox chemical, coupled proton transport, synthesis (ATP) reactions. Nuclear-encoded proteins are presented in grey whilst the chloroplast genome-encoded in white. Abbreviations: RC, reaction centre, OEC, oxygen-evolving complex; e − , electron; PQ, plastoquinone; H + , proton; FeS, iron-sulfur cluster; Fd, feridoxin; FNR, feridoxin-NADP-reductase.

Rupan_c03.indd 27 8/29/2012 5:39:01 PM


the first electron to the plastocyanin (PC) through the Rieske iron-sulfur protein and

cytochrome f. The second electron is passed through the cytochrome b 6 across the mem-

brane back to the stroma surface to reduce another oxidized plastoquinone. This peculiar

electron exchange, called the Q-cycle, increases the efficiency of proton translocation by

moving two additional H + for every two electrons, donated by water. Excitation of the PSI

reaction centre chlorophyll, P700, supports the continuation of the electron transfer chain

from PC to ferredoxin (Fd). From Fd, an electron can be donated either back to the

cytochrome b6/f complex, forming cyclic electron transfer to increase the amount of pro-

tons, translocated into lumen or to NADP reductase (FNR) to reduce NADP and finish the

linear electron transfer chain by forming NADPH. The accumulation of protons in lumen

leads to formation of a delta-pH across the membrane and an electrochemical potential,

which is used by ATP-synthase (ATPase) to form ATP from ADP and phosphate. ATPase

is another multisubunit complex that contains at least 20 polypeptides. In summary, of five

major types of photosynthetic membrane complexes only three carry pigments, light har-

vesting complexes, Photosystem I and Photosystem II core complexes. The structure and

functions, adaptations and assembly of these complexes will be a major focus of the chap-

ters that follow.

The reasons for grana formation, segregation of photosystems and their significance for

photosynthetic performance were always a focus of photosynthesis research. Stacked

thylakoids contain mainly photosystem II and its light harvesting antenna (LHCII) (see

also Figure 3.1 g and h). Unstacked membranes contain photosystem I with its own antenna,

LHCI, and ATPase. Cytochrome b6/f complexes tend to be located closer to the areas

where thylakoids begin to stack. Cytochrome b 6/ f and ATPase were also found in terminal

thylakoids of grana and ATPase was identified on the grana margins. To expect the high

efficiency of light energy utilization along the whole sequence of reactions one may look

for an existence of key forces or factors designed to integrate, conduct and make coherent

light absorption, excitation energy redistribution between photosystems, electron transfer

and proton translocation at conditions of special segregation between these components in

the photosynthetic membrane. It will later become evident that the central role in this

regulation is being played by the light harvesting antenna, the redox-state of the electron

transfer carriers and the delta-pH ( Δ pH) across the thylakoid membrane.

3.3 Oligomerization, Interactions and Mobility of the Photosynthetic Proteins: Enabling Functions and Adaptations

3.3.1 Oligomerization and Clustering of Photosynthetic Membrane Proteins

Although all five complexes of the photosynthetic membrane are built from relatively

light polypeptides, with the heaviest not exceeding 70 kDa (PsaA and PsaB), they them-

selves are very impressive nanoparticles. Photosystem II exists as dimer of reaction cen-

tre core complexes complemented by their own antenna complexes. Altogether they

form a stable so-called supercomplex structure of Photosytem II that can be isolated

biochemically or visualized using various electron microscopy approaches (see Chapter

Rupan_c03.indd 28 8/29/2012 5:39:01 PM


5 for details). The size of the dimeric PSII core is 430 kDa, whilst the size of the super-

complex is up to 1000 kDa and the area of ~300 nm 2 . However, PSII with full light har-

vesting complement could be almost twice larger. Full Phosystem I with its LHCI

antenna is a smaller particle with the molecular weight of about 600 kDa and area of

~200 nm 2 . Cytochrome b/f complex weighs approximately 220 kDa and has an area of

about 60 nm 2 . ATPase has molecular weight of ~400 kDa and its broadest part (CF 1 )

occupies area of ~80 nm 2 .

The reasons behind such macro-organization of the photosynthetic membrane complexes

are several. As was described earlier, the processes of photon energy transformation are

sequential and require a variety of cofactors and redox carriers organized in a particular

order that the events of light phase would flow in optimized way. At the start of the electron

transport, in PSII, a series of coordinated processes should take place including light

harvesting, focused transfer of trapped energy to the reaction centre, primary charge separation and stabilization, hence involving several electron carriers gradually remote

from the reaction centre to avoid back reaction. At the same time, the system of donation

of electrons to the PSII reaction centre that lost an electron is vitally important. This is a

number of interacting polypeptides that organize the water-splitting complex. The complex

must be associated with the reaction centre complex in order to deliver efficiently and

rhythmically electrons needed to maintain the electron transport through PSII. The RCII

electron donation-regaining cycle is called the turnover. The rate of this turnover is

important parameter that determines the productivity of the whole light phase of

photosynthesis. Naturally, the frequency of electron production from reaction centres will

determine the rate of NADP + reduction and proton translocation across the membrane

that will be used for ATP synthesis. PSII turnover rate is variable, depending on the

structural, environmental and metabolic factors. Maximum rate can be as high as 100

cycles per second, that is, 10 ms per cycle of electron removal from RCII and its re-reduction

by the donor side. The integration of light harvesting antenna within the PSII complex is

as important as its donor and acceptor side structures, since the antenna should supply and

as will emerge later regulate the photon energy supply to the reaction centre in a

sophisticated well-controlled manner. The latter is very important in order to maintain the

RCII in the optimal ‘working regime’, so to speak, its ‘rpm’ is within the reasonable safe

boundaries. Similar considerations apply to PSI.

The cytochrome b/f complex deals only with electrons and protons and ATPase with

protons and ADP, phosphate and ATP. Reduction and oxidation of plastoquinones, reduction

of mobile electron carrier plastocyanine in cytochrome b 6/ f complex are specially sepa-

rated processes that reside on different subunits. For ATPase a complex of proteins is

required to build the proton pumping engine, converting the gradient energy into the con-

formational energy of its subunits bringing ADP and phosphate together to form a high

energy phosphate bond of ATP. Only a multisubunit structure can carry such a task. Again,

the tight coordinated binding of polypeptides within these complexes is central for preci-

sion and directionality of carried reactions.

In supercomplexes of proteins, entropy is reduced and thermal energy can be utilized in a

more directional fashion to support and control reactions of the photon energy transformation.

Rupan_c03.indd 29 8/29/2012 5:39:02 PM


Hence, the protein environment can direct functions of various cofactors that are actually

involved in these processes. Summarizing the role of macrostructural organization of photo-

synthetic membrane complexes it is important to conclude that the key advantages of such

structure are:

a. lowering entropy;

b. binding and stabilizing the cofactors;

c. requirement for concerted and precize performance of the energy transformation reactions;

d. multifunctional performance of superstructures; and

e. fulfilment of the regulation requirement.

3.3.2 Protein Mobility

The photosynthetic membrane is a dynamic structure. The lipid phase is laterally mobile

despite of very high protein content. The five major photosynthetic membrane complexes

are not entirely stationary either. Figure 3.8 shows the results of the experiment which was

based on the bleaching of the probe fluorescence and observation of recovery (fluorescence

recovery after photobleaching, FRAP, see the next chapter for details). The bleaching is not

reversible, that can easily be tested on frozen or fixed with glutaraldehyde membranes. The

reason why fluorescence recovers is due to the lateral mobility of molecules of the probe.

In case of lipids, BODIPY (boron-dipyrromethene) probe has been used. This probe is

soluble in the lipid phase and therefore its diffusion is comparable with the diffusion rate

of lipids, that is indicative of how fluid the membrane is. As can be seen from the figure

mobility of BODIPY is rapid, takes place within seconds. It is important that even though

the membrane is crowded with proteins its lipid phase still remains very mobile. For the

photosynthetic membrane it is crucial, since the three of its complexes, PSII, Cyt b/f and

00 2 4 6 8 10

0

5

10

% recovery of protein%

rec

over

y of

lipi

d

15

20

25

50

75

100

125

Protein

Time, min

Lipid

Figure 3.8 Mobility of lipids and proteins in the photosynthetic membrane probed by the Fluorescence Recovery After Photobleaching (FRAP) method (for details see Chapter 4). Reproduced with permission from Johnson et al . © 2011 The American Society of Plant Biologists.

Rupan_c03.indd 30 8/29/2012 5:39:02 PM


PSI have to be connected to support the electron transport. PSII must be able to donate

electrons into the Cyt b/f complex. Plastoquinones are these freely diffusible transporters

in the photosynthetic membrane that maintain noncyclic as well as cyclic electron transport.

Plastoquinone diffusion rate is often a limiting step, a ‘bottleneck’ of the electron transport,

since, as was discussed earlier electron transport reactions which take place on the

complexes of photosystems and Cyt b/f complex are not limited by diffusion but occur

between cofactors rigidly positioned close to each other within those complexes. Proteins

in the photosynthetic membrane display much slower mobility. For example, Photosystem

II and LHCII antenna chlorophylls can be used as natural, intrinsic fluorescence probe in

the FRAP experiment (see Figure 3.8 ). It takes minutes for the fluorescence to recover.

Unlike in the case of BODIPY, the recovery is largely incomplete, meaning that some

components of PSII and LHCII are mobile within much slower time scale then 10 min or

not mobile at all. The protein concentration and associations into large oligomeric clusters

is likely to cause this. Examples of mobility of PSII and clustering of LHCII antenna will

be discussed in details a later chapter dealing with adaptations (Chapter 9). Indeed, protein

mobility in the photosynthetic membrane is essential not only for its functions but to enable

repair and replacement of proteins and their complexes essential for more long-term

changes involved in pant acclimation.

Lateral heterogeneity of the photosynthetic membrane is yet another feature that

illustrates that membrane is dynamic and the complexes are mobile. As shown in Figure 3.7 ,

PSII and LHCII antenna prefer grana stacks whilst PSI is located mainly in stromal,

unstacked thylakoids. It is simply impossible for ATPase, for example, to be localized in

the stacked grana regions simply because of the large structure of CF 1 component protruding

into the stroma. Such lateral redistribution is a process governed by charges on Photosystem

II and LHCII proteins. Indeed, as was mentioned above, magnesium and potassium cations

can compensate for the negative charges of stroma-exposed aminoacids of these complexes.

As a result, thermodynamically-driven regrouping of complexes would take place allowing

them to interact with each other as well as with the complexes positioned on different

thylakoids forming grana stacks. This is a well-reversible event in which LHCII is

particularly involved, since it is by far the major membrane complex accounting up to 50%

and even more of total photosynthetic membrane protein. It is important to point out that

the concentration of cations in stroma is a vital factor that controls grana formation.

Therefore, the metabolic homeostasis of stroma can be central in governing structure of

photosynthetic membranes and interactions between its complexes in vivo revealing the

way of regulation of the primary photosynthetic events by the chloroplast.

We have learned some basic principles of design of the photosynthetic membrane and

its proteins. It seems that the arrangement in two dimensions, on the surface, formation

of robust oligomeric states, crowdedness and interactions with lipids and essential

lipid-like molecules are important features that increase order, efficiency and enables

adaptations, hence flexibility of the membrane proteins. Therefore, the structural fea-

tures of the photosynthetic membrane allow running efficiently its multiple functions in

energy conversion, a remarkable example of vector chemistry that is typical for the

biological system.

Rupan_c03.indd 31 8/29/2012 5:39:02 PM


Reference

Johnson , M.P. , Goral , C.D.P. , Duffy , T.K. , et al . ( 2011 ) Photoprotective energy dissipation involves the reorganization of photosystem II light harvesting complexes in the grana membranes of higher plant chloroplasts . Plant Cell , 23 , 1468 – 1479 .

Bibliography

Anderson , J.M. , Chow , W.S. and Goodchild , D.J. ( 1988 ) Thylakoid membrane organisation in sun/shade acclimation . Australian Journal of Plant Physiology , 15 , 11 – 26 .

Barber , J. ( 1982 ) Influence of surface charges on thylakoid structure and function . Ann. Rev. Plant Physiol. , 33 , 261 – 295 .

Blankenship , R. ( 2002 ) Molecular Mechanisms of Photosynthesis . London : Blackwell Science . Clayton , R.K. ( 1980 ) Photosynthesis. Physical Mechanisms and Chemical Patterns . Cambridge :

Cambridge University Press . Dekker , J.P. and Boekema , E.J. ( 2005 ) Supramolecular organization of thylakoid membrane proteins

in green plants , Biochim. Biophys. Acta , 1706 , 12 – 39 . Hall , D.O. and Rao , K.K. ( 1995 ) Photosynthesis . Cambridge : Cambridge University Press . Hankamer , B. , Nield , J. , Zheleva , D. , et al . ( 1997 ) Isolation and biochemical characterisation of

monomeric and dimeric PSI1 complexes from spinach and their relevance to the organisation of photosystem II in vivo . Eur. J. Biochem ., 243 , 422 – 429 .

Heldt , H.-W. ( 2005 ) Plant Biochemistry . Burlington : Elsevier Academic Press . Walker , D. ( 1992 ) Energy, Plant, Man . Brighton : Oxygraphics .

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‘ By three methods we may learn wisdom: first, by reflection, which is noblest; second, by imitation, which is easiest; and third by experience, which is the bitterest .’

Confucius

There are far too many methods and approaches used in the research of the photosynthetic

membrane that we could cover in this book, or even several books. Here we aim to discuss the

major classes of the most popular methods that have been successfully applied in studies of the

photosynthetic membrane for a long time. All these can arbitrarily be divided into three groups:

1. Methods to analyse and manipulate composition of membrane components (biochemis-

try and molecular biology);

2. Methods to visualize membrane structures (structural biology);

3. Methods that study various functions of the photosynthetic membrane (analytical

approaches).

4.1 Biochemistry and Molecular Biology Approaches

4.1.1 Isolation of Chloroplasts and Subchloroplast Particles

Majority of biochemical approaches start from isolation of chloroplasts from leaves

(Figure 4.1 ). One of the challenges is to prepare intact chloroplasts that possess an

Popular Methods and Approaches to Study Composition, Structure

and Functions of the Photosynthetic Membrane

4

Rupan_c04.indd 33 8/29/2012 5:38:18 PM


unruptured envelope. Pioneering work of Robert Hill and later David Walker enabled

isolation of chloroplasts that were able to evolve oxygen and fix CO 2 to almost the same

rate as intact leaves. The advantage of using chloroplasts instead of leaves is that it is

easy to manipulate their functions with various inhibitors, uncouplers and cofactors that

can be added into the incubation cell. In addition the cell can be equipped with various

probes to test light harvesting, electron transfer and oxygen evolution functions (see

Section 4.3). The key factor in the preparation is time – the procedure must be per-

formed as quickly as possible. All is carried out at +4°C. Leaves are first ground within

a few seconds with a powerful blender, the homogenate filtered and centrifuged for less

than 1 min to pellet debris and then the second centrifugation step (~3000 g) is per-

formed through a density media, percoll, to separate more dense intact chloroplasts

from less dense broken chloroplasts and other components. Intact chloroplasts will

LeavesChloroplasts

Membranes

LHCII

Separated complexes

LHCIIb

LHCIIdLHCIIcLHCIIa

0.4

pH

4.2

4.6

0.7

Sucrose,M

PSII

PSI

Membrane fragments andsolubilised complexes

Grinding &centrifugation

Osmotic shock &centrifugation

+ Detergent &centrifugation

Ultracentrifugation onsucrose gradient

Isoelectric focusing(IEF)

Figure 4.1 Basic steps of isolation of the photosynthetic membrane complexes. (See Plate 4.1 in colour plate section.)

Rupan_c04.indd 34 8/29/2012 5:38:19 PM

Popular Methods and Approaches to Study Composition, Structure and Functions 35

appear as a pellet, which after washing and resuspending can be promptly used for

experiments. Good chloroplast prep from spinach can retain functions for up to 12 h,

sometimes even more.

The yield of intact chloroplasts is normally relatively low, therefore in order to get

more material for various functional and biochemical experiments a slightly different

procedure is used. It incorporates osmotic shock to break all chloroplasts and the second

centrifugation cycle time and rate are increased in order to pellet more ‘green’ material:

thylakoids. Naturally, the density media, percoll, is not required here. Thylakoids has

been a popular initial material for various biochemical preparations of the photosynthetic

membrane components. First, it aimed to isolate membranes enriched in photosystem

I or II, using the fact that they are located in spatially-separated thylakoids domains,

stroma and grana membranes, respectively. Jan Anderson first developed the procedure

that used detergent digitonin to gently remove stromal thylakoids from grana and sepa-

rate those by using several steps of ultracentrifugation, with a range of centrifugation

forces varying from 20 000 to 144 000 g. At lower speed larger particles, grana, are being

pelleted, whilst at the top rate, light membrane fragments, containing photosystem I will

be sedimented. The advantage of this procedure is that photosystems are embedded into

their natural environment, the thylakoid membrane. The disadvantage is that their sepa-

ration is not absolute. Grana fragments, in particular, were found to be contaminated

with photosystem I.

4.1.2 Isolation of Membrane Protein Complexes

In order to get better purification of photosystem II without compromising its functions, in

particular oxygen evolution Berthold, Babcock and Yocum ( 1981 ) developed widely used

procedure of preparation of so-called BBY particles. The procedure is based on isolation of

thylakoids, their incubation in the stacking medium to ensure maximum grana stacking

followed by treatment with non-ionic detergent Triton X100 on ice and subsequent cycles

of washing and centrifugation at ~30 000 g. As a result, membrane fragments containing

highly active photosystem II can be obtained with high yield.

Photosystem I digitonin particles contain significantly pure photosystem. However, fur-

ther isolation can be obtained by either solubilization of thylakoids of PSI fragments

by non-ionic detergents like n-dodecyl-β-D-maltoside or n-octyl-β-D-glucoside (see

Figure 3.4). Solubilized material can be applied to sucrose gradient of 0.2–1M and centri-

fuged overnight at 200 000 g. Photosystem I is a relatively stable complex and will appear

as a dense band at approx. 0.7 M of sucrose. In case of the solubilization of the whole thy-

lakoids, it is preferred to use unstacked membranes by incubation in the medium free from

cations (see Chapter 3). In this way not only photosystem I but photosystem II complexes

could be isolated on the sucrose gradient (Figure 4.1 ). All the LHCII antenna is completely

separated from photosystem II and the latter is represented as a reaction centre core dimer,

containing inner antenna complexes, CP43 and CP47. Solubilized thylakoid membranes of

membrane fragments could be also separated by gel filtration using, for example, Superdex

200 column (GE Healthcare) on a FPLC (Fast Protein Liquid Chromatography) system.

Gel filtration uses the principle of selection by molecular sizes. In fact it is filtration ‘in

Rupan_c04.indd 35 8/29/2012 5:38:20 PM


reverse’, since the fastest migrating are large particles and the slowest are small ones. The

advantage of this separation is that PSII supercomplexes and even PSII membrane frag-

ments containing highly ordered arrays of supercomplexes can be isolated. For this it is

required to use stacked thylakoids. Apparently magnesium is important not only for

stacking and segregation of photosystems but also to a full assembly of PSII with the

light harvesting antenna. Another way to prepare complexes of photosystems is to use

anion exchange chromatography. Here nonionic detergents must also be used. A linear

gradient of MgSO 4 is applied (5–25 mM) to run the separation of the DEAE

(Diethylaminoethyl cellulose) column. Molecular charges here are the key property that the

separation procedure is based upon.

An effective method that can be used to prepare various components of photosystems,

particularly antenna complexes, in their native state is isoelectric focusing ( IEF) (see

Figure 4.1 ). The principle of the method is based upon isoelectric point, pI. The soft IEF

gel contains ampholines. They are mixtures of amphoteric electrolytes or buffers that pro-

vide a continuous range of pH in an electric field. Proteins migrate in the field until their

charge is totally compensated at pH = pI. After that they stop migrating and narrow bands

of homogenous complexes can be visualized either by their green colour – all chlorophyll

binding proteins – or by using staining media in case of nonchlorophyll binding proteins.

For IEF it takes an overnight run and bands can be collected by spatula (since the IEF gel

is slurry) and eluted using special elution columns. This method proved to be effective,

preparative and of a high resolution (<0.1 pH units). Often to prepare photosystem com-

plexes of high purity a combination of isolation procedures is used. IEF often is followed

by additional purification step on the sucrose gradient, gel filtration or ion exchange chro-

matography. Naturally, the more purification steps are applied the lower the yield of

undamaged complexes.

An analytical way to separate thylakoid membrane complexes is to run nondenaturing

polyacrylamide gel (PAAG) electrophoresis. Sodium dodecyl sulfate (SDS), cholate or

n-dodecyl-β-D-maltoside were used for this method. It provides with relatively rapid (a

few hours) way to separate and analyse PSI, PSII and LHCII complexes and sometimes

called green electrophoresis, that was pioneered in Philip Thornber’s and Jan Anderson’s

laboratories. The disadvantage of this method is that the amounts of isolated complexes are

small and therefore it is hard to use them for further characterization work. Another disad-

vantage is that the gel media, polyacrylamide, and ionic detergents, SDS and cholate, often

affect the intactness of separated complexes stripping lipids and pigments from them.

Whilst photosystem II is relatively easy to disassemble with non-ionic detergents into

separate polypeptides, photosystem I is proved to be more resilient for such treatment.

Therefore, stronger nonionic detergents like Triton X100 or ionic detergents have to be

applied. Among the latter are SDS, cholate or zwitterionic detergents, that carry a positive

and a negative electrical charges at different locations within their molecule. Hence, for some

reason, PSI is a more robust structure then PSII. Non-pigmented complexes, like cytochrome

b6/f, plastocyanin and components of ATPase can also be prepared similarly to the three

‘green’ complexes typically using centrifugation and ion exchange chromatography.

Rupan_c04.indd 36 8/29/2012 5:38:20 PM


After the complexes of the thylakoid membrane are prepared, their polypeptide compo-

sition is normally analysed by classical SDS PAAG denaturing electrophoresis. For this

method procedures of removal of pigments and lipids are normally employed using organic

solvents like chloroform and ethanol, and proteins are broken into separate polypeptides

and undergo denaturing by special agents such as urea (used in molar concentrations) along

with a high temperature before running on a gel electrophoresis system. The polypeptides

molecular weight is estimated using standards: proteins of known molecular mass. However,

for certainty, the identity of proteins is estimated using Western blot procedure that is based

upon application of antibodies specific for polypeptides under analysis. Furthermore, in

some cases, the polypeptides of interest could be extracted from gel and subjected to

sequencing procedures for ultimate protein identification.

4.1.3 Analysis of Lipids and Pigments

Photosynthetic complexes ligands, lipids and pigments, can be analysed using various

chromatographic techniques, most notably analytical HPLC or high performance liquid

chromatography combined with the UV-Vis absorption detection. The introduction of

diode array detection enabled parallel collection of pigment spectra which greatly aids

quantification and localization of unknown compounds. Coupling HPLC with the mass-

spectrometer significantly enhances the system sensitivity and analysis of structural iso-

mers. The optical spectroscopic analysis of pigments is based on the fact that they normally

have specific absorption spectra. Chlorophylls and carotenoids of the photosynthetic mem-

brane can be first extracted from it with organic solvents (most typically acetone). Different

affinities of pigments to the column medium, determined by their hydrophobicity, is a key

factor that governs mobility of their molecules through the column or retention time in

often used reversed phase HPLC. There, the more polar pigments are eluted first, with the

shortest retention time; the more hydrophobic molecules will be eluted later since their

mobility will be reduced by their stronger tendency to interact with the column. A mix of

several solvents is usually used to gradually alter the mobile medium polarity (acetonitrile,

methanol, hexane) in order to achieve maximum resolution of individual pigments on col-

umn. Figure 4.2 displays a typical HPLC profile of all thylakoid membrane pigments. Each

pigment band corresponds to its maximum in absorption spectrum. The more oxygenated

and polar xanthophylls like neoxanthin and violaxanthin elute much faster than the less

polar lutein and zeaxanthin. In spite of the identical molecular mass, the latter two have

slightly different mobility because of configuration differences in the end-group orienta-

tion leading to the differences in the molecular polarity (will be explained in further chap-

ters). Isomerization of xanthophylls or partial chemical modification (oxidation) also alters

their mobility and is normally a sign of sample degradation induced by exposure to high

temperature, light, oxygen or chemical agents (for example, acids). The most hydrophobic

and less mobile is β-carotene, a true carotenoid that possesses no polar groups at all. The

HLPC system is normally calibrated using pigments of known concentration. This enables

to accurately quantify the molar amount of each pigments per leaf area, and so on. When

preparative amounts of pure pigments are required, thin layer chromatography (TLC) can

be used. This method does not require specialized equipment like HPLC, is relatively

Rupan_c04.indd 37 8/29/2012 5:38:20 PM


simple and inexpensive. It is often used to separate thylakoid membrane lipids, extracted

from chloroplasts using methanol/chloroform phase separation procedure. Since lipids do

not possess colour, their visualization on TLC plates is enabled using reagents like primu-

lin in combination with UV light illumination.

4.1.4 Protein Expression and Reconstitution In Vitro

The natural ability of proteins to refold under certain conditions from denatured state to

nearly native structure is utilized for production of large quantities of pure membrane

complexes, like LHCII, for example (Figure 4.3 ). Another advantage of using recombi-

nant proteins is that the point mutations can be easily introduced in order to probe func-

tions of particular aminoacids or even protein domains; for example, those involved in

cofactor binding, and so on. This procedure starts from expression of Lhcb genes of

LHCII apoprotein in E. coli : a popular bacterial model in molecular genetics. Expressed

protein is deposited in bacterial cell into the inclusion bodies. The latter are dissolved

using the buffer containing a strong ionic detergent lithium dodecylsulfate (LDS) and

eventually mixed with the pigment ethanolic extract at room temperature. The ionic deter-

gent is gradually replaced by milder, nonionic detergent, octylglucoside. After several

freeze-thaw cycles reconstituted complexes are separated from free pigments and proteins

Lut

0.035

0.030

0.025

0.020

0.015

Abs

orpt

ion,

rel

.

0.010

0.005

0.000

2.87

0

4.37

0

5.80

3

8.77

0

20.1

0320

.603

4.83

7

20.8

70

24.6

03

18.9

03

8.00

3

3.87

0

11.1

37

Neo

Vio

Zea

10.00

Time, minutes

20.00

Ant

Chl b

Chl a

β Car

Figure 4.2 HPLC analysis of the thylakoid membrane pigments. Neo, neoxanthin; Vio, violaxanthin; Ant, antheraxanthin; Lut, lutein; Zea, zeaxanthin; Chl b and a, chlorophylls b and a; b Car, b-carotene.

Rupan_c04.indd 38 8/29/2012 5:38:20 PM


using centrifugation. In case of LHCII that exists in trimeric form His-tagged protein is

used to enable immobilization onto a Ni-chelating column. There, the lipid, phosphatidyl-

glycerol, essential for trimerization is applied following release of LHCII from the column

with imidazol-containing buffer. Trimeric LHCII is separate from monomers using the

sucrose gradient centrifugation procedure described in Section 4.1.2 . The procedure can

yield a complex that is virtually identical to the isolated purified LHCII using IEF with

subsequent centrifugation or gel filtration. The intactness can be promptly tested using

absorption and circular dichroism spectroscopies (see Chapter 8 ).

4.1.5 Reconstitution of Membrane Proteins in Liposomes

Since the bilayer lipid membrane is a natural environment of the membrane protein it is

important to be able to incorporate isolated or reconstituted protein complexes into

liposomes: artificial membrane vesicles. Purified thylakoid lipids at composition and ratios

close to those of the native photosynthetic membrane (see Chapter 3) dissolved in chloro-

form are normally used for the procedure. The extract is dried under nitrogen to a thin film

in the reconstitution tube. Then a certain amount of buffer is added and the tube is sonicated

to form liposomes. A purified membrane complex, for example LHCII, is added to the

Plasmidmutagenesis

Expression vector:

pET-32b(5900bp)

E. colitransformation

Apoprotein

Pigments

Freeze-thaw

Reconstitutedcomplexes

Figure 4.3 Light harvesting protein expression and reconstitution with pigments. (Courtesy of Erica Belgio.)

Rupan_c04.indd 39 8/29/2012 5:38:20 PM


liposomes and mixed at certain protein/lipid ratio. Detergent needs to be removed by dialy-

sis or other relevant procedure followed by freeze and thaw cycles to induce formation of

the proteoliposomes that contain incorporated complexes. The latter are separated from the

rest by centrifugation on a ficoll gradient (a density medium used to separate cell orga-

nelles, etc.). The green band close to the top of gradient contains incorporated LHCII com-

plex that can be collected and concentrated using ultracentrifugation.

4.1.6 Mutagenesis and Transgenic Manipulations

Identification and expression of genes that encode various polypeptides as well as enzymes

engaged in biosynthesis of pigments, lipids and related photosynthetic membrane com-

pounds have become a popular pool of approaches aimed at identifying their functions in

the chain of energy transformation of primary photosynthesis. The traditional ‘forward

genetics’ approach is based upon the generation of mutations in a very large number of

seeds using radiation (neutron bombardment) or chemical agents like ethyl methanesul-

fonate, dimethyl sulfate or diethyl sulfate that modify DNA nucleotides. In many cases

mutations are not lethal and phenotypes can be screened in order to relate the lack of func-

tion to a modified/missing protein and identify a gene. Genetic mapping is done using PCR

(polymerase chain reaction), cDNA hybridization, sequencing and map-based cloning

tools. It is often possible to reach the saturation state, when all genes can be potentially

mutated in order to reveal a phenotype of interest. Whilst the traditional mutagenesis

begins with making mutants, the more recent approach, called ‘reverse genetics’ is based

on manipulation of a gene of interest in order to reveal a protein and function. This

approach is more incisive than ‘forward genetics’ since it allows the analyses of families

of genes with often redundant functions and performing combinations of mutations of

interest. One approach is based on the method of cosuppression (antisense) that introduces

RNA fragments that bind to mRNA blocking the translation process. However, the most

reliable and popular way to manipulate genes is based on T-DNA insertional mutagenesis.

It is based upon insertion of foreign DNA into the plant genome, hence a manipulation on

the transcription level. In Arabidopsis T-DNA of 5−25 kilobases can induce a significant

sometimes absolute and sustained gene disruption in almost all genes ( knock-out : KO).

Particular mutations can be identified using PCR. Both antisense and T-DNA manipula-

tions require application of the transformation procedure that uses Agrobacterium tumefa-ciens as a carrier of foreign gene constructs. This is a natural transformation model that

infects plant tissues and transfers a tumour-inducing (Ti) plasmid into the cell for integra-

tion into the nuclear genome and expression of bacterial genes that induce formation of

tumours in plant. For insertion mutagenesis, the T-DNA along with the genetic marker (for

example, kanamycin-resistance gene) is introduced into Ti plasmid replacing bacterial

genes using E.coli plasmid. The produced Agrobacterium strain is introduced into plant

using root or leaf dipping or vacuum infiltration techniques that deliver the bacterium into

plant tissues. A random way of T-DNA insertion into the plant genome as well as easily

identifiable tagging made it a very popular genetic approach of ‘reversed genetics’. On the

other hand, transformation system using Agrobacterium can also be used to ‘restore’ muta-

tions by supplementing plant with ‘working’ genes that have been corrupt by mutagenesis

Rupan_c04.indd 40 8/29/2012 5:38:21 PM


or manipulations. This method is useful not only to study gene functions but also to intro-

duce point-mutations in order to reveal the role of specific aminoacids in the produced

protein structure and functions in vivo .

Genetic manipulations described previously applied to photosystem I and II compo-

nents, pigments and proteins, significantly advanced and continue to further our knowledge

on their structure, functions in light harvesting, photoprotection and electron transport (see

the following chapters).

4.2 Visualization Techniques

4.2.1 Optical Microscopy

Optical microscopy was one of the first techniques that opened up a microworld of biology

and triggered great developments in microbiological sciences. It used light and a system of

optical lenses, that enabled the magnification of images of cells and their compartments

(see Figure 3.1c). It can work in light transmission as well as reflection modes. Figure 3.1(c)

shows an image of isolated chloroplasts with just visible dots of granae that are about

400 nm long and 300 nm thick. The image was taking with the highest aperture enabled by

application of emersion oil possessing high refraction index to increase the observation

angle. The resolution of optical microscopy is defined as:

,

2*d

NA

λ=

(4.1)

where d is resolution in nanometres; λ is the average wavelength of light used in micro-

scope (often 500 nm) and NA – numerical aperture ( NA = n sin( q ) ), where n is a medium

refraction index and q is one-half of the observation angle. In case of application of emer-

sion oil numerical amplitude is about 1.5. Therefore the maximum resolution at these con-

ditions can be less than 200 nm. Hence the granum size is at the limit of the conventional

optical microscopy resolution. Use of light with shorter wavelength or recently employed

lenses made of gallium phosphide enabling a nanosized focus enable to resolve objects the

size of ~100 nm.

Another way to increase optical resolution is to use fluorescence microscopy that is

based on observation of fluorescence light induced by laser and emitted from various

components of sample. Most common is confocal fluorescence microscopy, based upon

the use of very narrow illumination and eliminates the light which is not in focus and

undermines resolution. This microscopy just detects light from the parts of sample very

near the focal plane, ignoring the rest. Another advantage of the method is that specific

cell components can be visualized using fluorescence dyes or natural fluorescing (auto-

fluorescence) components. Figure 3.1(b) and Figure 3.2 display confocal images of leaf

cells that contain chloroplasts (in red). Here, the natural chlorophyll fluorescence was

used to produce an image. No other components of the cell are visualized, producing a

Rupan_c04.indd 41 8/29/2012 5:38:21 PM


clear unobstructed view of the chloroplast redistribution and even grana density in the

plant cell. Advanced fluorescence laser microscopies such as stimulated emission deple-

tion (STED) and total internal reflection (TIRF), have been employed recently to break the

optical resolution limit. STED uses the confocal scanning laser microscope with the sup-

plemental ‘fluorescence damping’ laser light to quickly switch-off the dye fluorescence

coming from the periphery of the excitation beam in order to narrow it to up to 10 nm

diameter, hence to decrease the optical resolution to this value. In TIRF microscope a very

thin layer of sample (~100 nm) is excited by the evanescent wave generated in a process

of reflection of a laser beam within the cover slip holding the sample, thus eliminating the

background fluorescence and enhancing the aperture. This technique is used not only for

studies of cell structure but also in research on single proteins immobilized in vitro (see

the following chapters).

4.2.1.1 Fluorescence Recovery After Photobleaching (FRAP)

Fluorescence Recovery After Photobleaching ( FRAP) is a popular method that uses confo-

cal laser scanning microscopy in order to study diffusion of membrane components, hence

dynamical aspect related to function. Chloroplast granae can be visualized by this method

(see Figures 3.1b and 3.2). Special buffers are used to immobilize isolated chloroplasts

onto a flat surface of a sample chamber. A gas laser line, often 488.0 nm, argon is used to

excite chlorophyll or external fluorescence dye (BODIPY) fluorescence. A set of filters is

used to define desired fluorescence spectral range. The same laser beam but much higher

intensity (a few orders of magnitude higher then scanning light) is briefly (a few seconds)

used to permanently bleach fluorescing molecules in one spot (one granum, for example)

following by reversing to the low intensity scanning mode to measure recovery of the fluo-

rescence of a beached area due to diffusion into it fluorescing molecules from the

unbleached thylakoid membranes. Low temperature or, indeed, cross-linkers will inhibit

membrane dynamics and therefore the bleach generated by FRAP technique will never be

restored. However, at ambient conditions it is possible to observe very rapid and complete

recovery of the lipid diffusion probe, BODIPY and partial, slower recovery of chlorophyll

fluorescence that belongs to photosystem and light harvesting complexes (Figure 3.8).

Obtained recovery kinetic data can be analysed in order to obtain diffusion coefficients for

lipids as well as pigment protein complexes in the photosynthetic membrane.

4.2.2 Electron Microscopy (EM)

4.2.2.1 Negative Staining EM

Electron microscopy (EM) is a high resolution method that uses electron instead of light

beam to study on the nanoscale level of cell structures including the photosynthetic mem-

brane. The high resolution of this type of spectroscopy is due to a very small wavelength

(<<1 Å) of fast moving electrons accelerated by high voltage in EM. The transmission

mode of EM is based upon the fact that electrons well penetrate the thin layers of biologi-

cal samples missing most of light nuclei of the major elements life matter is made of

(H, C, O, N) hence leaving little for the picture contrast. Therefore, the biological samples

Rupan_c04.indd 42 8/29/2012 5:38:21 PM


are often stained with salts of heavy metals, such as uranium (uranyl formate/uranyl ace-

tate). This approach is called negative staining procedure because the water-soluble stain

penetrates only hydrophilic regions of the membrane and its components, leaving hydro-

phobic ones unstained and therefore transparent for electrons. The whole photosynthetic

membrane can be visualized using thin section preparation method that includes fixation

by cross-linkers and incorporation of leaf/chloroplasts into the epoxy resin-based medium

followed by preparation of thin (100–200 nm) sections containing the fixed material using

a special knife, a microtome. Figure 3.1(f) shows an EM image of grana and stroma mem-

branes. The dark areas trace hydrophilic surfaces of membranes, where the stain was

deposited. Geometry of granum, number of thylakoids and their thickness can be estimated

from the image. In order to visualize individual photosynthetic membrane complexes, a

further preparation is required, often using detergent solubilization and FPLC technique to

isolate individual membrane fragments or protein complexes. Figure 4.4 (left) shows a

large grana thylakoid membrane fragment containing numerous PSII complexes.

Hydrophobic cores are seen as unstained rhomboid particles aligned in rows. A few loose

PSII particles are visible too. The resolution of this technique is not better than 2 nm, there-

fore the elements of PSII structure are poorly defined. To enhance it special image-process-

ing software is used to sort particles into different classes, align them and by summing up

the images within each class to reduce the level of noise. This process enhances the resolu-

tion up to five times. The topographic image on the right of the Figure 4.4 is a result of the

sum of thousands of images of PSII. The lightly stained dual core complex is positioned in

the centre of the particle that also contains four trimeric LHCII and all minor LHC antenna

complexes (details of this structure will be discussed in the following chapters).

Figure 4.4 Negative stain electron microscopy of the photosystem II membrane fragment isolated from Spinach thylakoids (left). Vertical arrows indicate the rows of PSII core complexes. Horizontal arrow indicates a single PSII particle. Bar is 50 nm. A single dimeric PSII core complex with attached two pairs of trimers and minor LHC complexes is presented on the right. Bar is 5 nm. Image on the right is provided courtesy of Egbert Boekema.

Rupan_c04.indd 43 8/29/2012 5:38:21 PM


4.2.2.2 Freeze-Fracture EM (FFEM)

Freeze-fracture (also known as freeze-etch) is an electron microscopy technique that uses

a specific method of sample preparation that is especially valid for examining biological

membranes, including the photosynthetic membranes of chloroplasts. The fresh protoplast

preparation (chloroplast or thyalkoid) has to be frozen rapidly (cryofixation) in special

sample holders first. The next step is to fracture the sample by breaking or using a special

knife (microtome) under liquid nitrogen or somewhat higher temperature. The frozen frac-

tured surface needs to be shadowed to enable contrast with evaporated heavy atoms of

platinum or gold at ~45° angle in vacuum followed by coating with carbon (evaporated

perpendicular to the average surface plane). This stabilizes the replica coating. A very frag-

ile heavy metal replica is removed from the sample at room temperature, usually by wash-

ing with detergent or strong alkaline solution (bleach) before application onto a fine grid

and viewing in transmission electron microscope. FFEM is most suitable for membrane

studies because the method very often splits membranes in half, thus revealing both of

internal membrane faces (Figure 4.5 ). The freeze-fracture technique splits the hydrophobic

core of the membrane bilayer into the exoplasmic and protoplasmic leaflets, allowing

information on the organization and dimensions of the proteins therein to be determined by

image analysis. The occurrence of such split can be explained by the fact the forces that

hold two lipid layers together (van der Waals) are the weakest forces in the cell (see Chapter

1). In a frozen state, the liquid phase is solidified by establishment of rigid water structures

with participation of the network of strong hydrogen bonds. Therefore it is easier to split

the membrane in half then remove it intact from the ice within the cell or chloroplast. In the

early 1970s membrane proteins were first identified in the FFEM images that allowed Singer

and Nicolson to propose their fluid-mosaic model of the biological membrane (Singer and

Nicolson, 1972 ). Figure 4.5 shows the diagram displaying positions of various membrane

proteins, some integral spanning some non-spanning as well as holes on an opposite half of

the membrane made by spanning proteins. Figures 3.1(g) and (h) and Figure 3.7 show typical

freeze-fracture images of grana membranes that accommodate PSII (EFs or exoplasm-facing

stacked membrane surface) and LHCII (PFs or protoplasm-facing stacked membrane surface)

complexes. The protoplasmic fracture face of the unstacked membranes (PFu) is

distinguished on the basis of its slightly larger asymmetric PSI particles. The complementary

E-Face

P-FaceFracture plane

Membrane proteins

Figure 4.5 The principles of sample preparation for freeze-fracture electron microscopy. E- and P-faces are the exoplasm (space outside of an organelle) and protoplasm (space insight an organelle) facing surfaces of the split membrane.

Rupan_c04.indd 44 8/29/2012 5:38:22 PM


exoplasmic fracture face of the unstacked membranes (EFu) is largely smooth and marked

by generally more widely spaced (low concentration) PSII particles. An example of analy-

sis of multiple protein particles is displayed in Figure 4.6 . Special image-analysing soft-

ware automatically identifies proteins, determines distances between them, highlights

protein clusters, and so on. The resolution of FFEM is not as high as the EM that uses

negative staining procedure, only ~5 nm. This is largely determined by the fact that not the

sample but its heavy metal replica that is being studied. The thickness of the replica masks

fine features of the membrane proteins leading to the decrease in resolution.

4.2.3 Atomic Force Microscopy (AFM)

All previously described methods of electron microscopy have one disadvantage, the state

of a sample is largely artificial: cross-linkers and stains are used to condition components

of the photosynthetic membrane. AFM is an alternative high resolution (up to 5 Å) tech-

nique that in principle can be applied to the intact photosynthetic membrane or at least

membrane fragments containing hundreds of proteins at ambient temperature and free

from any artificial chemical agents. AFM visualizes the topography of an object via a

microscopic probe (cantilever) with a very thin tip of molecular, nanoscale, sharpness

(Figure 4.7 ). To map the object the cantilever scans over it. During this scanning the probe

constantly or intermittently (oscillating mode) interacts via atomic interactive forces with

the sample molecules. The sharper the tip, the smaller is the interaction area between it and

the sample and hence the better resolution could be achieved. Since the force strength

depends upon a distance between the probe and the sample, the feedback loop enabled by

piezo-actuator attached to the probe (similar to the vinyl record playing machines) ensures

always the same interaction force by constantly adjusting the vertical position of the canti-

lever over the un-even membrane surface (protruding proteins and their clusters with lipids,

etc.). As a result a topographic scan is produced revealing various structural elements of the

membrane surface. The accuracy of the tip position can reach 10 Å. These days AFM has

been developed into a multipurpose approach that can detect apart for the membrane sur-

face image various important biophysical parameters. Therefore it has become possible to

study structural and functional properties of biological membranes in parallel. Results of

AFM applications in the photosynthetic membrane research will be increasingly in focus

for years to come.

4.2.4 Crystallography Methods

4.2.4.1 X-ray Crystallography

Electron microscopy methods described above can zoom into the nanoworld with minimum

dimension of around of 1 nm, a more than two orders of magnitude improvement in

comparison to the optical microscopy. The sizes of membrane proteins and their complexes

with pigments and often oligomeric forms vary within 5–25 nm. As far as the groupings

and redistribution of membrane proteins are concerned as well as some crude imaging of

shapes, 1 nm resolution is sufficient. However, the knowledge of domain structure, helixes,

Rupan_c04.indd 45 8/29/2012 5:38:23 PM

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Rupan_c04.indd 46 8/29/2012 5:38:23 PM


cofactors and finally specific aminoacids remains out of reach of microscopies. In order to

see them one has to go into the atomic resolution, that is a few Ångstroms: another order

of magnitude down from nanometre and a window open for studies of life chemistry and

physics. Protein crystallography methods are designed to obtain a resolution of up to 1 Å.

With this resolution contours of single atoms (electron density maps) could be obtained,

hence distinguishing atoms of carbon from oxygen, and so on in the 3D state of a protein.

X-ray crystallography is the method of choice, where possible. The short wavelength of

X-rays enables us to see very fine molecular features as small as single atoms. But

membrane proteins are generally harder to crystallize than water soluble proteins. The first

membrane protein complex to have its structure solved by X-ray crystallography at high

resolution was the photosynthetic reaction centre of purple bacteria. For this work Michel,

Deisenhofer and Huber were awarded Nobel Prize for Chemistry in 1988 (Deisenhofer

et al ., 1985). Knowledge of the protein structure details is believed to be essential for

understanding its function. One disadvantage of X-ray crystallography, however, is that a

membrane protein is taken from its environment, membrane, and its structure may not

correspond to the structure of a native complex and is inevitably a static one. The method

is also not free from a few difficulties. The most serious problem is to obtain good quality

crystals that will diffract at the highest resolution. If the resolution is only about 4 Å it

Figure 4.7 The principles of Atomic Force Microscopy (AFM). A laser beam is reflected from the cantilever monitoring its position. The controller receives this information and drives the piezo-actuator to compensate for the cantilever deflection in vertical direction, feedback compensation. Information on the sample microscopic landscape is processed by computer to generate an image.

Rupan_c04.indd 47 8/29/2012 5:38:25 PM


would be almost impossible to unambiguously identify the aminoacid side chains.

However, alpha helixes can be identified with this resolution. At a resolution of about 2.5 Å

most of the atoms could be identified and water and other components like ions can be

seen. Resolution depends on the regularity of crystal as well as its stability during the

measurement of the diffraction picture. Crystallization is an entropy-driven process. The

build-up of order within the crystal is gained at the expense of increase in entropy of the

medium; molecules of solvent, detergent, and so on are being expelled from the proteins

that build crystal. Factors like pH, temperature, composition of crystallization medium can

strongly affect the quality of crystals. Therefore multiple crystallization trials, often using

automated multisampling equipment are required for establishing optimum crystallization

conditions. In an X-ray diffractometer crystals are subjected to an X-ray beam where its

scattering occurs. This scattering represents actually a multiple reflection process within

the regularly ordered proteins and their multiple structural elements. Reflected rays often

diffract and as a result a diffraction pattern is registered on the screen (photographic film

or camera). The interpretation of this pattern is based on the Bragg’s law that links

parameters of the structural elements of the diffracting protein to the complex diffraction

picture. This pattern is processed using Fourier transformation analysis in order to obtain

protein 3D electron density map. The next step is to identify or fit a molecule to particular

element in the map. Here, the resolution (the map quality) is of paramount importance,

since the higher resolution the more unambiguously certain density can be assigned. With

lesser resolution the molecular contours are less clearly defined leading to the increase in

uncertainty of assignment.

4.2.4.2 Electron Diffraction

Fast electrons also exhibit wave behaviour with the wavelength even shorter than X-rays.

They can be used in studies of 2D protein crystals. The latter are much easier to obtain in

comparison to 3D crystals. Membrane proteins tend to form lamellar sheets where they

often are organized into ordered arrays that can be obtained at high rate under certain crys-

tallization conditions. The resolution is not quite as good as X-ray crystallography (>3 Å),

but is often used for membrane proteins that cannot be crystallized in three-dimensions and

in order to obtain at least some information about helixes, domain structure, number of

cofactors, and so on. The data analysis is based upon Fourier transformation similar to the

analysis of X-ray diffraction patterns.

4.3 Function Probing Methods

Although biochemistry and visualization approaches provide fundamental information

about the membrane components, like identification, composition and structure, function

probing methods are a large group of approaches that aim to address various mechanistic

aspects of the photosynthetic membrane works. This is by far the largest class of methods

used to study the photosynthetic membrane functions with majority of them suitable for in vivo research. Many of the methods applicable for functional studies are based on the use

of various spectroscopies to gently analytically probe photosynthetic membrane functions.

Rupan_c04.indd 48 8/29/2012 5:38:26 PM


The popularity of spectroscopic methods in photosynthesis research can be explained by

the fact that primary photosynthetic processes are based upon the use of light by photosys-

tems to trigger the chain of energy conserving events (see Figure 1.1). As can be seen in

Chapter 5, the particles that perform the primary photosynthetic energy transformations are

electrons of pigments and electron transport carriers. The reactions they are involved into

are by nature photobiological and therefore accompanied by various photobiological pro-

cesses, like absorption, fluorescence, phosphorescence, anisotropy, and so on that can be

studied by optical spectroscopy.

4.3.1 Absorption-Based Approaches

Processes of light harvesting, excitation energy migration among pigments and electron

transfer between carriers are often followed by spectral changes in absorption. The time-

scale of these changes varies depending upon the type of reaction. Visible-UV light-based

absorption methods are used broadly in the photosynthesis research in steady-state and

kinetic modes of measurements. Light absorption spectroscopy can be defined as an analy-

sis of light absorption by matter as a function of wavelength (λ). The central principle of

any optical absorption spectroscopy is based upon the Beer-Lambert law. Figure 4.8 shows

the scheme of light absorption within the sample of a depth L. Propagating within the sam-

ple light is gradually absorbed and the intensity of transmitted beam, I , is less than the

intensity of incident one, I 0 . The decrease in the incident light intensity due to absorption

within an infinitely thin section of sample, dx , is proportional to the intensity of probing

light and the concentration of sample molecules, C :

,dI I C dxα= − ⋅ ⋅ ⋅⋅ ⋅ ⋅

(4.2)

or

,

dICdx

Iα= −

(4.3)

I=0 dx

Solution oflight absorber

Transmitted light, IInsident light, Io

Figure 4.8 Principles of optical absorption spectroscopy. Derivation of the Beer-Lambert law.

Rupan_c04.indd 49 8/29/2012 5:38:27 PM


where a is proportionality coefficient (constant). Now both parts of the equation can be

integrated resulting in:

0

.I

ln CxI

α⎛ ⎞

= −⎜ ⎟⎝ ⎠

(4.4)

The left part of Equation 4.4 is linearly proportional to sample concentration and sample

depth/thickness as well as a constant α . In spectroscopy natural logarithm is normally

replaced by common logarithm:

010log ,Cx OD

Iε⎛ ⎞ = =⎜ ⎟⎝ ⎠

I

(4.5)

where 2.303ε α= is called molar extinction coefficient with dimensions (M −1 cm −1 ). OD is called optical density or absorption and is dimensionless. Basically, OD indicated the

power of magnitude of reduction in incident light intensity as a result of absorption within

a sample. If OD = 1 , this means that the transmitted light intensity for a sample is only 10%

of the incident one. If OD is 2, then only 1% of incident light is left after absorption. OD is a convenient and simple way for estimation of molecular concentration. The extinction

coefficient is dependent upon the wavelength of absorbed light. Normally it is given at the

maximum of absorption spectrum, λεmax

. Figure 4.9 displays a basic scheme of a simple

spectrophotometer. Light from the light source (normally tungsten halogen, deuterium or

mercury lamp) is directed into a monochromator. The latter contains a prism of diffraction

grating that split light into various relatively monochromatic colours or wavelengths,

which are sequentially directed upon a sample and the transmitted light intensity is detected

by the detector (photodiode, photomultiplier, CCD: charge coupled device, camera, etc.).

The analyser registers intensity for all probed wavelengths. However, in order to do so, I 0

needs to be registered too. For this often a second channel (dual beam regime) is used

where a reference cuvette, containing a solvent or buffer in which the sample molecules

were dissolved, is placed. The analyser automatically calculates a common logarithm from

the I 0 / I corresponding to each wavelength used and reconstructs an OD spectrum.

Figure 4.9 Basic scheme of an absorption spectrometer.

Rupan_c04.indd 50 8/29/2012 5:38:28 PM


In reality, the most conventional spectrophotometers are limited by the sensitivity of the

detectors and the presence of so-called stray light that originates from the incident light

bypassing the sample without being absorbed (reflection from the cuvette walls, scatter-

ing, etc.). This causes underestimation of the true OD and heavy distortion of absorption

spectrum due to the loss of linearity between OD and concentration. Therefore, it is gener-

ally recommended to use the sample at somewhat low concentration, where OD varies

from 0.3 to 0.8, for the most of spectrophotometers. Photosynthetic membranes as well as

isolated membrane complexes are relatively large particles that inevitably cause light scat-

tering during absorption measurements. This can lead to spectral distortion. Normally, OD

becomes overestimated throughout the absorption spectrum, since the escape of light from

the cuvette and photomultiplier is interpreted by the analyser as a lost transmitted light. To

correct this artefact, the sample is often positioned closely to the detector, light diffuser

(opal glass) is used to equally scatter light in the sample and reference cuvettes and, finally

and most effectively, light scattering particles (latex or milk) are added to the reference

cuvette to obtain there levels of scattering equal to those of the sample. Poorly transparent

or non-transparent samples, like leaves, are often measured in reflectance regime. The

sample is being probed by light at 45°with reflected light detection at the same angle. The

reflected light intensity is taken instead of transmitted one in calculation of OD.

Among the most common applications of absorption spectrophotometry in photosynthe-

sis research is the determination of chlorophyll concentration. First, pigments need to be

extracted using organic solvents, most often acetone. Extract OD is measured at maxima of

chlorophyll a and b absorption and concentrations of these pigments are calculated using a

formula specific for the solvent, since the extinction coefficient varies depending upon the

solvent type. Chlorophyll a/b (Chl a/b ) ratio is a popular parameter, characteristic of the

membrane or isolated pigment-protein composition. Particular care needs to be taken to

preserve stability of pigment extracts, since light and acid exposure can easily damage

chlorophylls leading to underestimation of their concentration. Total carotenoid content

can also be calculated using this method.

Absorption spectral measurements of isolated pigment-proteins can be useful in their

preliminary identification and characterization. In order to improve signal to noise ratio,

procedures like Fourier filtration can be applied. Figure 4.10 compares spectra of a multi-

component system before (original) and after (smoothed) such filtration. For gaining more

information about number of components and their accurate maxima positions, calculation

of the second or fourth derivative is often used. The maxima of pigment spectra are much

better resolved in derivative mode, since the spectral bandwidth for example in the second

derivative spectrum is almost two times narrower than that in the original one. Figure 4.10

shows that the second derivative spectrum revealed at least five clearly defined maxima,

whilst the original spectrum showed only one clear maximum and two bands appeared as

shoulders. Narrowing bands in spectroscopy is of paramount importance, since it can allow

analysis of spectra of very complex systems, like isolated pigment-protein complexes or

even whole photosynthetic membranes, that contain many types of pigments and their

forms originating from the same pigment bound into different microenvironments/binding

pockets (for details see Chapter 6). Another way to increase the spectral resolution is to

Rupan_c04.indd 51 8/29/2012 5:38:30 PM


cool the sample to the temperature of liquid nitrogen (77 K) or even liquid helium (4 K).

Measurements of absorption spectra at these temperatures are complicated by the issues of

sample transparency that can be addressed by using glycerol-based sample media to pre-

vent water crystallization resulting in very strong light scattering.

Absorption spectroscopy is also widely applied as a tool to monitor certain photosynthetic

electron transfer reactions. In conditions where electron acceptors are missing from the

thylakoid or BBY particles preparations, it is possible to support/restore the electron trans-

port chain by using artificially added electron acceptors such as dichlorophenol indophenol

(DCIP), potassium ferricyanide, ferredoxin, and so on. Their absorption spectra are very

sensitive to the redox state. For example, reduced DCIP is colourless whilst the oxidized is

blue. The electron transfer rate can simply be measured by the rate of DCIP absorption

change caused by illumination.

Information about spectra of components of electron transfer chain can be obtained

using differential absorption spectroscopy. There, the sample cuvette contains sample that

undergoes treatment with chemical reagents, temperature or, indeed, light. The reference

cuvette contains, instead of buffer, an untreated sample. The difference spectrum treated-

minus-untreated is automatically acquired. Often it reveals very specific spectral informa-

tion on some minor components whose spectra are heavily masked by the spectra of

chlorophylls, for example. This approach allowed producing spectra of cytochromes and

reaction centre chlorophylls.

Original

Smoothed

2nd derivative

Figure 4.10 Basic spectral analysis using methods of digital filtration and second derivatives.

Rupan_c04.indd 52 8/29/2012 5:38:30 PM


Another insightful pool of absorption methodology applications comes from kinetic spec-

troscopy. Since photosynthesis is a sequence of events that gradually stabilize and slow-down

energy given by light (see Chapter 1), it is possible to register these events using various time

windows and wavelengths of detection as well as the level of intactness of the sample. Energy

transfer and trapping events in photosystems’ pigment ensamples are accompanied by tran-

sient alterations in absorption of pigments. Pigments that receive energy and are excited can-

not absorb light until they undergo de-excitation. Excited states leave from nano- to

picoseconds or even shorter. Therefore, the accompanied light-induced absorption changes

have to be recorded very rapidly by specialized equipment using streak-cameras, laser pulses,

delay circuits, and so on. The method is generally called pump-probe spectroscopy with

various evolved modifications. It can be used to study isolated pigment protein complexes,

photosystems, photosynthetic membrane fragments or, indeed, chloroplasts and even leaves.

It helps greatly to understand primary photosynthetic photophysical and photochemical

events and explain high efficiency of energy conversion in the photosystems. For slower reac-

tions of electron transport ranging from submicroseconds (in photosystems) to milliseconds

(cytochromes, etc.) flash-photolysis equipment is normally used. It is based on using repeti-

tive flashes of light from xenon lamp strong enough to saturate all reaction centres by one

flash with duration of a few microseconds. The same equipment can be used to measure so-

called electrochromic shift that originate from the altered absorption of some carotenoids as

a result of a build-up of electric field across the photosynthetic membrane. The field is build

due to electron and coupled proton transfer across the membrane. This technique became

popular for estimations of the extent of the transmembrane proton gradient in chloroplasts

and leaves. Finally, the slowest light-triggered absorption changes that take place during the

light phase of photosynthesis in the membrane associated with enzymatic pigment intercon-

version and establishment of the photoprotective energy dissipative state (xanthophyll cycle,

see Chapter 9) can be monitored on the timescale of seconds and minutes. Very often the

kinetic absorption measurements use a dual wavelength regime that involves a reference light

of a certain wavelength, where no absorption change is expected to take place, to be used in

the same light path as a measuring beam. This way of referencing allows performance of

more stable and accurate measurements compared to the dual beam configuration.

So far, we have mentioned various absorption spectroscopy approaches that provide infor-

mation on the type and concentration of pigments. However, absorption spectroscopy has

much broader potential. Applications of spectroscopies that use polarized light enable to

study pigment architecture in protein complexes and the membrane, their mutual orientation,

interactions with each other and protein as well as their molecular conformation. Two meth-

ods of polarization spectroscopy are often used in photosynthesis research; linear, LD, and

circular, CD, dichroism absorption spectroscopies. For LD, light is polarized using a special

crystal in one plane of its electric field oscillation (see Chapter 5). Membranes of isolated

pigmented complexes need to be oriented by magnetic field or using various mechanic meth-

ods (stretching in gel or brushing onto the glass slide). LD uses light polarized either in paral-

lel as well as perpendicular to the sample alignment. A sample absorption of light beams with

these two orientations is analysed to produce LD that is normally proportional to Δ A = A − A ⊥ ,

where A and A ⊥ is absorption of light oriented in parallel and perpendicular planes relatively

to the sample molecules alignment. The LD spectrum of the photosynthetic membrane

Rupan_c04.indd 53 8/29/2012 5:38:30 PM


or isolated pigment-protein complexes contains a number of bands that belong to carotenoids

and chlorophylls indicating relative orientation of these pigments as well as possible close

energetic interactions (excitonic coupling). CD uses light that is helically oriented producing

a spiral of its electric field propagation in space. It can spiral clockwise (right) or anticlock-

wise (left). In this case, circular dichroism is a difference between absorption of left and right

circularly polarized lights: Δ A = A L − A

R . For CD, a sample does not have to be oriented. The

CD spectrum of isolated chlorophylls or carotenoids is normally very weak and featureless.

On the contrary, the CD spectrum of protein-bound pigments is very strong. This is because

binding causes conformational strain and distortion of a pigment molecule that reflects in the

increase in its optical activity. Interactions between pigments can also result in appearance of

strong CD spectrum with one positive and one negative band (conservative change, no gain/

loss in absorption) of excitonic origin. Modern polarization absorption spectrophotometers

can easily combine LD and CD modes of absorption measurements that can work in spectral

or kinetic modes at a range of sample temperatures.

Infrared absorption spectroscopy (IR) is another absorption-based method that utilizes

infrared light in order to probe energies of molecular vibrations. They are an order of

magnitude weaker than those of electronic energy and can be potentially useful in

identification of molecular species (for example pigments) and their interactions with

environment (hydrogen bonding, etc.) as well as studies of membrane protein secondary

structure. Kinetic IR spectroscopy is used in photosynthesis research to probe structural

changes in cofactors and their environment as a result of photochemical reactions of

electron transport. Use of interferometers instead of monochromators enables better

stability and promptness of spectral recording. Employment of attenuated total reflection

(ATR) sample compartments makes it possible to do measurements on very small sample

volumes, semidried films and use of nontransparent samples.

4.3.2 Raman Spectroscopy

Raman spectroscopy is becoming a popular tool in photosynthesis research. It is a method

which uses Raman or combinational scattering effect that occurs when the monochromatic

laser light is absorbed by sample. The majority of light photons that are not absorbed by a

molecule tend to reflect from it producing light scattering or more accurately elastic

Rayleigh scattering. The scattered light has the same wavelength as incident light. However,

there is a probability that incident light will interact with vibrations of sample molecules

resulting in alterations (decrease or increase) in the energy of incident light photons

(Figure 4.11 ). The shift in energy of exciting light is due to molecular dynamics originating

from constant vibrational and rotational motions (see Chapter 5). Therefore the Raman

shift gives information about the energy of these modes in the sample molecules. In fact,

Raman spectroscopy is similar but complementary to the IR spectroscopy, since it reveals

vibrations normally not seen by IR. The information gained by Raman spectroscopy is very

useful in identification of molecular species, pigments; for example, their environment,

conformation, and so on. Raman scattering is normally very weak. However, if the wave-

length of the incident light matches that of the electronic (UV-Vis) absorption spectrum the

probability of Raman scattering dramatically increases, sometimes up to one million times.

Rupan_c04.indd 54 8/29/2012 5:38:30 PM


Besides, resonance conditions excite only one or a very few molecular species that absorbed

the incident light wavelength, therefore this method can be highly selective suitable to

study particular types of molecules in very complex mixtures with others. Particularly use-

ful is resonance Raman in studies of carotenoids: secondary light harvesting pigments.

Carotenoids unlike chlorophylls are nonfluorescent, therefore they cannot be studied by

fluorescence spectroscopy. Fortunately they are very strong Raman scatterers, more effec-

tive then chlorophylls. Therefore, the majority of important parameters of the photosyn-

thetic carotenoids have been obtained using resonance Raman spectroscopy (for details see

Chapter 8). One of the major disadvantages of resonance Raman spectroscopy is the often

strong fluorescence excited by incident light that is overwhelming for the sensitive Raman

emission detectors. In addition, photodegradation or photoisomerization of the sample can

take place since the energy of incident light is normally high. This, however, can be par-

tially overcome by cooling of the sample to the liquid nitrogen temperature (77 K).

4.3.3 Fluorescence-Based Approaches

Fluorescence, a type of emission of light that takes place after absorption of photon by a

pigment molecule, has always been popular in research of the photosynthetic membrane.

It counts a number of classes of approaches to studying the photosynthetic membrane

Laser line(Rayleigh scattering)

Raman scattering

Energies of molecular vibrations0

ν4

ν3

ν2

ν1

Figure 4.11 Spectra of scattering of incident laser line (488 nm) – Rayleigh and carotenoid in solvent – resonance Raman. Energies of individual intramolecular vibrations are determined by the extent of the shift of Raman spectrum from the energy (wavelength) of scattered incident laser light (Rayleigh).

Rupan_c04.indd 55 8/29/2012 5:38:30 PM


and its components and is being rapidly developed to gain novel insights into its structure

and functions. Fluorescence of chlorophylls, the main photosynthetic pigments, provides

vital information about their variety, energy transfer between them, interactions with the

environment, efficiency, adaptations of the photosynthetic membrane to environmental

conditions, and so on. One of the reasons for the popularity of fluorescence applications

could be that it reveals the fate of light energy absorption by the membrane as well as

reflects the state of electron transport and even ATP synthesis and carbon metabolism.

4.3.3.1 Fluorescence Spectroscopy

Fluorescence compete with a number of nonradiative processes (see Chapter 5 for details)

that usually make fluorescence light coming out of sample much weaker than the light that

is absorbed ( I a = I

0 − I ) (see Figure 4.8 ). Hence, fluorescence depends first of all upon the

sample absorption, that include concentration, the absorption path and molar extinction as

well as upon the efficiency/rate of competitive nonradiative processes. Fluorescence

intensity can be expressed as:

,f

ff nr

kI I

k kα

⎛ ⎞= ⎜ ⎟+⎝ ⎠

(4.6)

where I a is the intensity of absorbed light or incident light minus transmitted light inten-

sity; k f is the fluorescence rate constant and k

nr is the sum of all nonradiative rate constants.

+( ( ))f f nrk k k is known as the fluorescence yield ( f f ) and is always less than unity. The

chlorophyll fluorescence yield in solvents approaches 0.33 or 33%, meaning that one out

of three absorbed photons on average will be reemitted as a fluorescence photon. However,

it is usually hard to capture all fluorescence photons coming out of a sample upon absorp-

tion, since they are being emitted in various directions, in a similar (but not identical)

manner to light scattering (Figure 4.12 ). Therefore, the majority of fluorescence spectral

measurements taken at steady state give only relative fluorescence intensity. Figure 4.12

shows components of a typical spectrofluorimeter. Light defined by excitation channel

monochromators excites fluorescence at a certain wavelength, λ exc

. Fluorescence is emit-

ted by the sample in different directions with some delivered to emission monochromator,

that disperses it into a range of wavelengths in order to generate a range of intensities,

detected and processed by the detector and analyser to obtain a fluorescence spectrum.

A range of special sample compartments can be used in order to adopt various types of

samples: liquid solutions of isolated chloroplasts or complexes, leaf fragments or indeed

the whole nondetached leaves. In addition, an optical cryostat can be placed in the sample

compartment in order to enable measurements at cryogenic temperatures, most often 77 K

or 4 K. Some cryostats enable to use a broad range of temperatures from room to cryogenic

for studies of thermodynamic properties of samples. The emission monochromator wave-

length can be fixed at maximum of fluorescence spectrum whilst excitation monochroma-

tor could scan a range of wavelengths in order to produce excitation fluorescence spectrum .

Rupan_c04.indd 56 8/29/2012 5:38:31 PM


This type of spectrum in case of a homogenous pigment system has a shape of I a or I 0 − I – a

spectrum of absorbed excitation light intensity. It is not to be confused with a logarithmic

absorption spectrum, log ( I 0 / I ), if one wants to compare excitation and absorption spectra.

Indeed, ( I − I 0 ) amplitude is not a linear function to pigment concentration. Therefore the

shape of excitation fluorescence spectrum will differ at different concentration of a pig-

ment. It will become increasingly broader with the concentration increase. In case of chlo-

rophyll it is better to undertake the measurements at reasonably low concentration,

corresponding to OD = 0.1 or even lower. If excitation fluorescence spectra of various sam-

ples need to be compared, it is essential to make sure that the optical densities of all samples

are equal. Figure 4.13 shows a scheme explaining the principle of excitation fluorescence

spectroscopy. This is the case of multicomponent system with energy transfer between pig-

ments directed towards the red-most pigment, called terminal emitter. Fluorescence of this

emitter is detected at the wavelength of its maximum. Excitation light is scanned in the

short wavelength region throughout the whole absorption spectrum of the system. The

resulting excitation fluorescence spectrum can be expressed as:

( ) ( )0

1

( ) * ( ) ( ) ,n

i ii

F f k I Iλ λ λ=

= −∑

(4.7)

Light source

Detector

Analyser

Emissionmonochromator

Excitationmonochromator

Sample cuvette

lf

lo

Figure 4.12 Basic scheme of a spectrofluorimeter.

Rupan_c04.indd 57 8/29/2012 5:38:31 PM


where f is a proportionality coefficient k i is the energy transfer efficiency coefficient of

i component (varies from 0 to 1), ( I 0 ( l ) − I

i ( l )) is a reversed transmission spectrum of an

i component. If k i < 1 it means that there are some energy losses during its transfer to the

terminal emitter and the corresponding excitation fluorescence spectrum amplitude will be

decreased in comparison to the reversed transmission spectrum of the system.

Fluorescence can be excited by linearly polarized light and detected through a polar-

izer oriented either in parallel or perpendicular direction to the excitation light polariza-

tion plane to probe fluorescence polarization, P , that is equal to Δ I / I , where Δ I = I − I ⊥ and I = I + I ⊥ where I and I ⊥ are fluorescence intensities at parallel and perpendicular ori-

entation of polarized relatively to the polarization plane of excitation light. More often

than P, another polarization parameter, fluorescence anisotropy, is used: ⊥= Δ +/ ( 2 )r I I I

If the excited pigment molecule changes orientation/conformation or passes energy to

another pigment P will decrease. The fluorescence anisotropy spectroscopy of photosyn-

thetic membrane or pigment protein complexes is used to identify pigments involved in

energy transfer or those which are uncoupled due to the damage or their specific origins

and functions.

4.3.3.2 Time-Resolved Fluorescence

The formula for fluorescence quantum yield expressed in terms of rate constants. This is

derived from the monoexponential type of decay of excited molecular state. This results

from the solution of a differential equation:

= − +( ) ,f nr

dnk k n

dt (4.8)

Ene

rgy

ki (I0 – Ii(λ))

λ

Absorption

Fluorescence

Figure 4.13 Principles of fluorescence excitation spectroscopy. Fluorescence excitation spectrum of a multicomponent system connected with the energy transfer (down arrows) directed to the terminal emitter (the lowest absorption level) the fluorescence of which is detected.

Rupan_c04.indd 58 8/29/2012 5:38:32 PM


where n 0 and n are number of excited molecules at the time of absorption of light and at

time t , respectively. The minus sign indicates decay in n with time. Regrouping and integra-

tion of this equation gives us:

( )

0 ,f nrk k tn n e

− +=

(4.9)

where ( k f + k

nr ) correspond to an exponential decay rate constant. Therefore the yield can be

expressed as:

0

,f f

ff nr

k

k k

τφ

τ= =

+

(4.10)

where t f = 1/( k f + k nr

) and t 0 = 1/ k

f is the intrinsic radiative lifetime. This lifetime corresponds

to the lifetime of the excited state in the absence of all nonradiative deactivation processes,

when the yield is unity. The measured fluorescence lifetime is always shorter than the

intrinsic lifetime due to the presence of a combination of various nonradiative decays (see

Chapter 5). For chlorophyll a the intrinsic lifetime is around 15 ns, whilst the maximum

measured fluorescence lifetime is about 5 ns giving the yield of about 0.33, as was

mentioned previously. Measurement of fluorescence decay is therefore a unique method

that enables to estimate absolute fluorescence yield and obtain kinetic information about

processes of energy transfer, trapping and de-excitation within the photosynthetic pigments,

pigment-protein complexes and the membrane as a whole.

Phase fluorometers, time-correlated single photon counting (TCSPC) or streak camera

devises are used to measure chlorophyll fluorescence lifetimes. The basic scheme of a

TCSPC lifetime fluorometer is similar to that depicted in the Figure 4.12 . However, for

excitation pulsed lasers are used. In addition the system counts every fluorescence photon

and estimates the time taken for it to arrive onto the detector, basically working as a very

fast timer, that times photons arriving at various times and reconstructs the decay curve

using thousands of counts. Many components of a lifetime fluorometer are specifically

adopted to enable the fast response of the system. A TCSPC machine possesses average

time resolution of about 50 ps (0.05 ns). However, the analytical software enables to

increase this to about 10 ps (0.01), which is reasonably good for analysis of fluorescence

lifetimes in the range of 0.1–5 ns. Normally temporal resolution is also complemented by

spectral resolution so that decay-associated spectra of complex samples possessing several

chlorophyll-emitting bands can be calculated.

4.3.3.3 Pulse Amplitude Modulated (PAM) Fluorescence

Nowadays, chlorophyll fluorescence has become one of the most rapid and nondestructive

methods for efficient assessment of photosynthetic membrane performance in plants in situ

popular among various kinds of plant researchers. At ambient temperature the fluorescence

of the intact photosynthetic membrane originates mainly from PSII, maximum at

Rupan_c04.indd 59 8/29/2012 5:38:33 PM


680–685 nm with a small contribution from PSI around 720 nm. Hence, the both photosys-

tems fluoresce at far-red edge of the visible spectrum. This is due to a very different

arrangement of antenna pigments in these photosystems (see Chapter 6). The fluorescence

intensity is proportional to the yield that is defined by the ratio of radiative and the sum of

all de-excitation constants, as defined in the previous paragraph (in Section 4.3.3.2). Apart

from fluorescence there are two more major channels of de-excitation in PSII, nonradiative

dissipation into heat (rate constant k d ) and photochemistry (rate constant k

P ) due to the reac-

tion centre activity – a major event starting the chain of photosynthetic reactions

(Figure 4.14 ).

Hence the chlorophyll fluorescence yield of the photosynthetic membrane with ‘ working’

PSII reaction centres can be expressed as:

,( )

f

ff d p

k

k k kφ =

+ +

(4.11)

Yields of nonradiative heat dissipation and photochemical activity of PSII reaction centres

can be expressed by analogy as:

,( )

dd

f d p

k

k k kφ =

+ +

(4.12)

HeatLHA

RC

Fluorescence

Photochemistry

ML

PLAL

Figure 4.14 Principles of pulse amplitude modulated fluorescence measurements.

Rupan_c04.indd 60 8/29/2012 5:38:34 PM


and

,( )

p

PSIIf d p

k

k k kφ =

+ +

(4.13)

It is easy to figure out if one can control one of the three dissipation channels it would be

possible to find out the yields of the other two. Fortunately, the photochemistry can be

blocked by chemicals, like DCMU, or by using a light flash of very high intensity. Both

factors cause the closure of RCII that manifests in reduction of the Q A acceptor that causes

chlorophyll fluorescence to rise. Saturating pulses are more frequently used in fluorescence

analysis since the invention of the pulse amplitude modulated fluorimeter. This devise uses

pulse-triggered lock-in amplifier system that registers fluorescence only induced by pulsed

excitation light. Any other fluorescence induced by continuous or pulsed light of different

frequency and duration, no matter how bright it is, will not be registered by this fluorime-

ter: it will simply not respond to any signal but pulsed light of the programmed frequency

and duration. This is the basic principle of PAM fluorimetry. Measuring pulsed light is set

normally at very low intensity (<1 μ M m −2 s −1 ) that is barely visible by eye. Figure 4.15

shows a typical fluorescence trace obtained from a leaf measurement.

The level of fluorescence induced by the measuring beam is very low, however, it is

not equal to zero no matter how low is the exciting light intensity. This is because the

photochemical channel rate constant is not infinitely large. This fluorescence level is called F o

and reflects the state when all PSII centres are open and consume the energy of the measuring

beam. In order to obtain the idea how much larger is the photochemical channel in comparison

to the fluorescence one a 1 s very bright light pulse (>5000 μ M m −2 s −1 ) is applied. This light

competitively saturates all PSII reaction centres with energy, so they are not anymore able to

utilize the energy of the measuring beam light. Hence, the k P relatively to the measuring light

becomes zero. The fluorescence rises to a maximum level called F m . The fluorescence yields of

F 0 and F

m can be expressed as:

( )0

f

F

f d P

k

k k kφ =

+ +

(4.14)

and

( )m

f

Ff d

k

k kφ =

+

(4.15)

It is easy to find out from these two equations by simple algebra transformations that:

0( )

m

m

F F

PSIIF

φ φφ

φ−

=

(4.16)

Rupan_c04.indd 61 8/29/2012 5:38:35 PM


Since mF mKFφ = and

0 0F KFφ = , where K is the proportionality constant that links relative

fluorescence intensity with the fluorescence yield and is always the same regardless of the

state of photochemistry or other processes taking place in PSII it is possible to express f PSII

as:

( )0,

m vPSII

m m

F F F

F Fφ

−= =

(4.17)

where F v = F

m − F

0 .

F v / F

m is the first fundamental parameter that defines the quantum yield of PSII photo-

chemistry. It is normally 0.8 or 80%, a very efficient light energy conversion into the energy

of electron moving into the electron transport chain. PAM fluorimetry is therefore a unique

method that allows calculation of fundamental absolute parameters like yields without

measuring absolute fluorescence yield. A third type of light, called actinic light, can also be

Pulses1 min

Time

Fm’

Fm

Fm’’

Fs

Pulse

Flu

ores

cenc

e

Actinic lighton

Actinic lightoff

Measuring light

Figure 4.15 Typical chlorophyll fluorescence induction curve of dark-adapted leaves measured using a PAM fluorimeter. First, fluorescence is excited by very weak, pulsed light (<1 m Mm −2 s −1 and frequency of 1.6 kHz), called ML (measuring light), to monitor Fo, the fluorescence state of antenna, when reaction centres are open for excitation energy delivery. Then 1 s pulse of saturating light (PL, 5000 m Mm −2 s −1 ) was applied in parallel to close all reaction centres. This almost immediately brought the fluorescence level to its maximum, Fm. Therefore the quenching by open reaction centres, given as (Fm-Fo)/Fm is used to quantify the photochemical quenching, which indicates how efficiently light is being utilized. However, it does not account for all of the quenching observed. Indeed, in light close to saturating for electron transport (AL, here intensity was ~1000 m M.s −1 .m −2 ), quenching by reaction centres is reduced almost to zero, yet there can be large amounts of other type of quenching. Such quenching is therefore called non-photochemical quenching and refers to the difference between the initial, dark-adapted Fm and that recorded after a period of illumination, Fm′ This quenching has been termed variously qN or NPQ.

Rupan_c04.indd 62 8/29/2012 5:38:36 PM


applied to actually observe the fluorescence induction trace (Figure 4.15 ). This light is

continuous and of somewhat moderate intensity, chosen to keep some reaction centres free

from saturation. On top of this light, bright light pulses can be applied to see the level of

maximum fluorescence. What is remarkable, first of all, is that this maximum level during

actinic light illumination is at first very high, reaching Fm level in the dark but then becomes

to decline, sometimes very steeply and within a few minutes or longer stabilizes at some-

what low level called F m ′. The yield of F

m ′ is expressed as:

,( )m

f

Ff d D

k

k k kφ =

+ +

(4.18)

where k D is a rate constant of the nonradiative process that causes F

m level decline. This

lowering of Fm induced by continuous light it termed nonphotochemical quenching, qN . The yield of this process, f

D , can be calculated as

,( )

DD

f d D

k

k k kφ =

+ +

(4.19)

where k D is a transient nonradiative dissipation constant, or induced non-photochemical

fluorescence quenching constant. Often f D is referred to as qN and expressed via

flu orescence levels:

( )m m

m

F FqN

F

− ′=

(4.20)

The nonphotochemical quenching is also quantified as ( F m − F

m ′)/ F

m ′. This calculation

simply corresponds to the ratio k D /( k

f + k

d ), which basically compares the rate of

nonphotochemical quenching process to the sum of all other rates at the conditions when

all PSII reaction centres are closed and is referred to as NPQ . Interestingly that the level of

fluorescence under actinic light without pulses is somewhat lower ( F s ) and indicates at least

partial contribution of reaction centre activity to the fluorescence quenching. The latter is

called photochemical quenching, qP , and calculated as:

0

m s

m

F FqP

F F

′ +=

′ + ′

(4.21)

In the dark, this quenching equals 1. The PSII yield at actinic light relates to qP via

following formula:

.vPSII

m

FqP

Fφ =

(4.22)

Rupan_c04.indd 63 8/29/2012 5:38:37 PM


Figure 4.14 also shows that the light-induced fluorescence decline relaxes upon removal of

actinic light, so that at least the major part of F m is restored after some 5–10 min of the dark.

This relatively quickly reversible qN component was originally called energy−dependent quenching , qE . Nowadays it is often related to a measure of ‘induced dissipation’ or

‘photoprotective energy dissipation’. The yield of qE is calculated as:

m m

m

F FqE

F

″ ′=

′�

(4.23)

The corresponding NPQ parameter for the reversible quenching will be

/ /r m m m mNPQ F F F F ″′= � . Chapter 9 will discuss causes, role, origins and mechanisms

of nonphotochemical quenching in detail. The dynamic fluorescence behaviour presented

in Figure 4.15 was first discovered by Kautsky 80 years ago and still fascinates scientists

and reflects a chain of events of electron and proton transport that are being sequentially

born in the photosynthetic membrane upon absorption of light (Kautsky and Hirsch, 1931 ).

PAM fluorimetry is a quickly-developing technique that became well-accepted and used

in laboratories, in field experiments on land and under water. PAM cameras and micro-

scopes have been produced to measure photosynthetic performance of individual plant

seedlings, cells and even chloroplasts. It is now being combined with the measurements of

oxygen evolution during illumination, proton gradient formation (see the next paragraph)

and other processes that are triggered by the photosynthetic activity of the membrane.

Combined with the time-resolved fluorescence spectroscopy that measures absolute fluo-

rescence yields (see the previous paragraph) it became one of the most popular and insight-

ful tools that can study in situ the photosynthetic membrane efficiency and dynamic

mechanisms of adaptations to light environment.

4.3.3.4 ΔpH Measurements using Fluorescence Probes

Externally added fluorescence probes can be used for spectral measurements on the

photosynthetic membrane to monitor its functions. One of them is the dye called

9- aminoacrydine (9-aa). It cannot be used on intact or detached leaves but chloroplast suspen-

sions of a certain volume. Its absorption maximum is around 350 nm and fluorescence is

around 400 nm. Therefore it is not in overlap with the far-red fluorescence of photosystems

chlorophyll and can actually be monitored in parallel with chlorophyll fluorescence induction

of chloroplasts. Indeed, the measurements are made in kinetic mode with detection in 400–

420 nm region defined by an optical interference filter. This amine in protonated form redis-

tributes itself across the membrane. The protonation takes place as a result of ΔpH formation

resulting from the photosynthetic electron transport (see Chapter 3). It takes normally around

5–15 s for complete establishment of the proton gradient, a much slower than some of the

electron transfer rates described earlier. Therefore, it does not require a sophisticated equip-

ment to perform these measurements. 9-aa is fluorescent in both, protonated and nonproto-

nated forms. However, once diffused in the thylakoid lumen, the space of which is very small,

it easily concentrates and becomes nonfluorescent as a result of so-called concentration

Rupan_c04.indd 64 8/29/2012 5:38:39 PM


quenching. Therefore, the measured quenching of 9-aa induced by ΔpH formation is simply

due to the removal of fluorescent 9-aa from the stroma into the lumen where its fluorescence

vanishes completely. Light-induced 9-aa quenching can be calculated as:

9 ,

i q

aai

F Fq

F=�

�

(4.24)

where Fi and F

q are fluorescence before illumination (and therefore ΔpH establishment)

and after formation of ΔpH, respectively. This quenching is normally noticeable, around

50% or even more. The numerical difference (will be negative) between lumen and stromal

pH is expressed as:

( )− Δ

⎡ ⎤ ⎛ ⎞= +⎢ ⎥ ⎜ ⎟⎝ ⎠⎢ ⎥⎣ ⎦9

9

,1

aa

aa

q VpH log log

vq�

��

(4.25)

where V and v are the total volumes of the chloroplast-containing suspension and the inter-

nal volume of all thylakoids, that is lumen, of the whole sample. Whilst is it relatively easy

to monitor 9-aa quenching, provided it is used at right concentration (~1 mM) and ratio to

the chlorophyll concentration of the sample (~1 : 40) , it is often hard to estimate correctly

to know the lumen volume, v . There are, however, a few reports that provide values of the

lumen volume that are often used in calculations of ΔpH using formula (Equation 4.25 ).

ΔpH induced in the dark by hydrolysis of ATP can also be monitored using the 9-aa method.

The accurate estimation of the lumen volume is the most controversial issue of the 9-aa

method making some to look for alternative approaches to measure proton gradient.

References

Berthold , D.A. , Babcock , G.T. and Yocum , C.F. ( 1981 ) A highly resolved, oxygen-evolving photosystem II preparation from spinach thylakoid membranes: EPR and electron-transport properties . FEBS Lett. , 134 , 231 – 234 .

Deisenhofer , J. , Epp , O. , Miki , K. et al . ( 1985 ) Structure of the protein subunits in the photosynthetic reaction centre of Rhodopseudomonas viridis at 3Å resolution . Nature , 318 ( 6047 ), 618 – 662 .

Kautsky , H. and Hirsch , A. ( 1931 ) Neue Versuche zur Kohlensäureassimilation , Naturwissenschaften , 19 , 964 – 964 .

Singer , S.J. and Nicolson , G.L. ( 1972 ) The Fluid Mosaic Model of the Structure of Cell Membranes . Science , 175 ( 4023 ), 720 – 731 .

Bibliography

Berg , J.M. , Tymoczko , J.L. and Stryer , L. ( 2012 ) Biochemistry . Basingstoke : W.H. Freeman and Company .

Boekema , E.J. , Folea , M. and Kouril , R. ( 2009 ) Single particle electron microscopy . Photosynth. Res ., 102 , 189 – 196 .

Rupan_c04.indd 65 8/29/2012 5:38:40 PM


Blankenship , R. ( 2002 ) Molecular Mechanisms of Photosynthesis . London : Blackwell Science . Branden , C. and Tooze , J. ( 1991 ) Introduction to Protein Structure . New York and London : Garland

Publishing . Carpentier , R. (ed.) ( 2004 ) Photosynthesis research protocols . Methods in Molecular Biology , 274 ,

1064 – 3745 , New Jersey : Humana Press . Clayton , R.K. ( 1970 ) Light and Living Matter, Volume 1: The Physical Part . New York : McGraw-Hill . Hall , D.O. and Rao , K.K. ( 1995 ) Photosynthesis . Cambridge : Cambridge University Press . Hipkins , M.F. and Baker , N.R. (eds.) ( 1986 ) Photosynthesis: Energy Transduction: A Practical

Approach. Practical Approach Series . Oxdord : IRL Press Limited . Kirchhoff , H. , Haferkamp , S. , Allen , J.F. et al . ( 2008 ) Protein diffusion and macromolecular crowd-

ing in thylakoid membranes . Plant Physiol. , 146 , 1571 – 1578 . Krause , G.H . and Weis , E . ( 1991 ) Chlorophyll fluorescence in photosynthesis: the basics . Ann. Rev.

Plant Pysiol. Plant Mol. Biol ., 42 , 313349 . Krysan , P.JU ., Young , J.C. and Sussman , M.R. ( 1999 ) T-DNA as an insertional mutagen in

Arabidopsis . The Plant Cell , 11 , 2283 – 2290 . Miller , D. ( 2008 ) AFM: a nanotool in membrane biology . Biochemistry , 47 , 7986 – 7998 . Moya , I. , Silvestri , M. , Vallon , O. et al . ( 2001 ) Time-resolved fluorescence analysis of the photosys-

tem II antenna proteins in detergent micelles and liposomes . Biochemistry , 40 , 12552 – 12561 . Østergaard , L. and Yanofsky , M.F. ( 2004 ) Establishing gene function by mutagenesis in Arabidopsis

thaliana . The Plant Journal , 39 , 682 – 696 . Paulsen , H ., Rümler , U . and Rüdiger , W . ( 1990 ) Reconstitution of pigment-containing complexes

from light-harvesting chlorophyll a/b-binding protein overexpressed in E. col i. Planta , 181 , 204 – 211 .

Robert , B. , Horton , P. , Pascal , A.A. and Ruban , A.V. ( 2004 ) Insights into the molecular dynamics of plant light−harvesting proteins in vivo . Trends in Plant Science , 9 , 385 – 390 .

Salinas , J. and Sanchez-Serrano , J.J. ( 2006 ) Me thods in Molecular Biology, Volume 323, Arabidopsis Protocols , 2nd edn . Totowa, NJ : Humana Press Inc .

Serdyuk , I.N. , Zaccai , N.R. and Zaccai , J. ( 2007 ) Methods in Molecular Biophysics . Cambridge : Cambridge University Press .

Schreiber , U. ( 1986 ) Detection of rapid induction kinetics with a new type of high frequency modu-lated chlorophyll fluorometer . Photosynth. Res. , 9 , 261 – 272 .

Staehelin , L.A. ( 2003 ) Chloroplast structure: from chlorophyll granules to supra−molecular architecture of thylakoid membranes . Photosynth. Res. , 76 , 185 – 196 .

Rupan_c04.indd 66 8/29/2012 5:38:41 PM




‘ The hardest thing of all is to find a black cat in a dark room, especially if there is no cat .’ Confucius

5.1 The Nature of Light

In Chapter 1 defining life we mentioned that essential for it was the availability of unlimited

amount of a special sort of energy, that was easily transported in space, basically wireless,

and was specific . What kind of energy is it? Nature has chosen light. It travels with the fast-

est speed attainable in our Universe, it carries energy that can be ‘addressed’ to a certain

receiver, a type of molecule, hence the specificity of light energy. Some molecules are

totally indifferent to visible light, and therefore transparent, like water, the major constitu-

ent of life. On the other hand, certain molecules are coloured, hence interact directly with

visible light, as it will be explained later. Light is therefore the most convenient form of

energy for the life in the universe, a source of building up negative entropy: order, an essen-

tial attribute of all life forms.

The nature of light presented scientists with a serious challenge at the beginning of

the twentieth century contributing to the so-called crisis in physics resulted in emergence

of new fundamental disciplines explaining the nature of matter and light – the special and

Primary Processes of the Light Phase of Photosynthesis: Principles of Light Harvesting

in Antennae

5

Rupan_c05.indd 67 8/29/2012 5:37:44 PM


general theories of relativity, quantum mechanics, quantum electrodynamics and the

physics of elementary particles – all nano- and even attoworld sciences. Whilst the mechanics

of the macroworld created by Newton assumed that particles and waves are different

manifestations of nature, particle coordinates, the movement trajectory and energy are pre-

cisely predictable obeying the relatively simple laws. These properties of our macroworld

are perfectly in agreement with our daily perception of matter around us. However, at the

beginning of the twentieth century physicists started to understand that classical Newtonian

mechanics was failing to describe the motions of the nanoworld; electrons, atoms, molecules

and most of all, light. The appearance of theoretical explanation of the nature of light

preceded the development of physical understanding of the nature of electricity and

magnetism. At first glance two independent, invisible force fields known by scientists

for some time and later found to be fundamentally connected in Faraday’s discovery of

electromagnetic induction were justified by Maxwell in his theoretical work. Electromagnetic

waves originate from an oscillating electric dipole. These harmonic oscillations of electric

field are followed by oscillations in magnetic field oriented perpendicularly to the former.

Both propagate in space with the speed of light (Figure 5.1 ):

,c λυ= (5.1)

where c is a speed of light (~3 . 10 8 m/c) l is wavelength (as in Figure 5.1 ) and u is a

frequency of field oscillations.

Maxwell’s approach provided fundamental grounds for the future that explained various

electromagnetic wave phenomena observed in nature. Now we can construct a scale of a

large range of electromagnetic waves, keeping the speed of light constant and varying

wavelength/frequency as shown in Figure 5.2 .

The magnitudes of the wavelengths span from hundreds of metres, for radio waves

to fractions of nanometres for ionizing radiation. The frequency changes greatly too: from

Megahertz for radio waves to Exahertz for X-rays. This is a colossal span of nearly 15

orders of magnitude, a remarkable feature of electromagnetic waves. Visible light inhabits

λ– Wavelenth

Oscillatingdipole

L, distance

M+

–

E

Figure 5.1 Electromagnetic wave: origin and propagation. M and E, magnetic and electric field amplitudes.

Rupan_c05.indd 68 8/29/2012 5:37:47 PM

Primary Processes of the Light Phase of Photosynthesis 69

a very narrow region in this scale of electromagnetic waves, just 400–700 nm. The fact

that we see these waves is due to a photophysical reaction they cause in the pigments of

the retina. Luckily we see only a narrow electromagnetic wave span, otherwise we

could be easily lost in the world of visible, radio, micro, infrared and UV radiation. But

photosynthetic organisms use only this region simply because its source, the Sun, has the

maximum of available for Earth electromagnetic emission in the visible region. Figure 5.3

shows the spectrum of solar radiation at the surface of Earth.

Figure 5.2 The spectrum of electromagnetic waves in nature and the place of visible light in it.

500 1000

Wavelength, nm

Ligh

t int

ensi

ty, r

el.

Figure 5.3 The spectrum of sunlight on land surface in an open field. (Courtesy of Eric Murchie.)

Rupan_c05.indd 69 8/29/2012 5:37:47 PM


The maximum of Sun’s spectrum is positioned around 500 nm. Numerous troughs can

be seen particularly on the right side of the spectrum. They are Fraunhofer lines, absorption

due to atmospheric water and oxygen. This spectrum of radiation is all that feeds our

biosphere with energy. Visible light fluctuates with a colossal frequency of about 0.6 peta-

Hertz or 6.0 × 10 14 fluctuations per second! What kind of dipole fluctuates with such agility?

The answer is electrons of atoms and molecules. Hence, light radiation originates from the

nanoworld as the result of very fast movements of tiny particles. These particles, electrons,

can not only emit light but also can be set by light into oscillation of the same frequency.

This is a process of resonance similar to that observed in the macroworld of sound and

mechanical waves. One vibrating tuning fork can set off the vibrations of another, closely

positioned, provided they have the same resonant frequency. Light as an electromagnetic

wave is a carrier of a certain amount of energy that can be absorbed by matter and reemitted

giving it back into space. Hence the light-matter interaction is a process of constant energy

transformation, of moving charges and electromagnetic waves propagating in space. This

exchange of energy was thoroughly investigated by Planck ( 1900 ), who proposed that

it must be discontinuous, occurring in discrete portions of energy – quanta. Hence the

radiation energy and its interaction with matter should be quantized. Planck suggested that

electrons of atoms possess only a certain number of fixed energy levels, some of which can

be resonant with the energy of light:

υ= ,E nh (5.2)

where h is a constant (Planck’s constant, = 6.62 . 10 −34 J . s), n = 0, 1, 2 … The quanta of light

energy are called photons. Their energy depends only upon their frequency. The colour of

light defines its energy. Therefore, blue light (~450 nm) has higher energy than the red light

(~650 nm). The intensity of light is simply determined by the amplitude of waves or number

of photons. Hence, the colour determines the resonance energy required to excite certain

dipoles within the matter, whilst intensity of light means quantity of photons that carry this

fixed level of energy. Here we enter the special nature of light radiation, wave-particle

duality. It comes from the origins of light from the subnanoworld of atoms, where tiny

particle dimensions, colossal speeds of electron movements disobey the laws of classical

mechanics so that it becomes impossible to track precisely their coordinates and their

energies. They possess ‘fuzzy’ trajectories that can only be described by probability

functions. Some experiments like photoelectric effect and response of light to gravity

confirmed the particle properties of light. However, diffraction of light is a typical property

of wave. This conflict of light properties was addressed by De Broglie ( 1922 , 1923 ) who

proposed that such dualism is an inherent property of all matter, including the macroworld

we live in, implying an interesting generalization of the laws of fundamental physics in

macro- and nanoworlds. Indeed, also we consider ourselves as reasonably particular, not

wave-like creatures; we possess properties of waves, with the wavelength of:

,h

pλ =

(5.3)

Rupan_c05.indd 70 8/29/2012 5:37:48 PM


where p is the mechanical momentum ( p = mass × velocity ). According to this formula,

particles that move fast should possess shorter wavelength. Such formula is the basis of

the electron microscopy that by accelerating electrons decreases their wavelength far

beyond the optical diffraction limit enabling to achieve very high special resolution and

see various details of nanostructures including those of the photosynthetic membrane

(see Chapter 4).

5.2 Absorption of Light by Molecules

The movements of electrons within atoms and molecules are also described by a quantum

theory that provided a fundamental ground for understanding the evolution and relation-

ships between all elements in nature. Schrödinger ( 1925 , 1926 ) devised an equation that

could define the states of electrons in atoms. The solution of this equation provides a set of

discrete energy levels that correspond to electrons located on different atomic orbitals,

number and types of which are unique to an each element of matter, like hydrogen, carbon,

oxygen, and so on. Negatively charged electrons move in their orbitals around positively

charged nuclei. There is no emission of electromagnetic radiation until they change an

orbital, where they will have different energy value. Difference in the energy values gives

an electromagnetic radiation that can be emitted by atom. In a similar manner, if the

electromagnetic wave (light) will interact with electron and cause it to change the orbital,

the energy of this radiation will be absorbed and be equal to the difference between energy

levels after and before the act of absorption. Figure 5.4 shows two discrete orbitals of an

atom with energies E 1 and E

2 . The light with energy h u can potentially be absorbed if its

energy is equal to E 2 − E

1 – conditions of resonance. The change in the dipole moment

during transition is the second requirement of absorption to occur. Hence a certain degree

of asymmetry is required.

Absorption spectrum of a simple single transition will look, in most cases, like a line of

intensity corresponding to a single energy value. The line will have a finite width owing to the

poorly defined energy of the excited state that originates from the energy-time uncertainty

Energy

Inte

nsityExcited

state

Emission

Absorption

Groundstate

hν = E2–E1

hn

E1

E2

Figure 5.4 Light is a highly selective form of energy. On the right: the schematic presentation of the absorption spectrum corresponding to the scheme.

Rupan_c05.indd 71 8/29/2012 5:37:49 PM


principle, proposed by Heisenberg ( 1927 ). It states that it is impossible to be equally precise

in determination of the energy of a particle and time it possess this energy (electron, etc.). The

longer the particle lives at a certain energy level, the more accurately this energy can be

defined. The shorter the particle stays in a certain energy state, the less defined will be its

energy. For a molecule, a system of various molecular orbitals exists that are occupied by

electrons. The higher occupied molecular orbital (HOMO) accommodates two electrons that

are optically active or react to the electromagnetic field of radiation. Upon absorption, excited

electron will occupy lowest unoccupied molecular orbital (LUMO). However, in addition to

electronic energy ( E el .

), energy of vibrations ( E vib .

) and rotations ( E rot .

) of atoms within a mol-

ecule will be added to the electronic energy highly enriching the spectrum of energy levels in

the ground (HOMO) and excited (LUMO) molecular states (Figure 5.5 ). In addition, the

translational movement on a molecule in space ( Ε trans .

) will also add to the total molecular

energy:

= + + +m el. vib. rot. trans.E E E E E

(5.4)

As a result, the absorption spectrum will be complex, much broader than the line of atomic

absorption spectrum. Interaction of motion of electrons with the electromagnetic fluctua-

tions in the immediate, condensed matter environment (vibrations and polarization by the

solvent) can result in homogenous broadening of absorption spectrum. In the biological

environment, water-based or hydrophobic membrane interior absorption spectrum will also

be broadened by variations in electronic energy due to variability of microenvironment of

individual molecules (inhomogeneous broadening) or existence of a number of different

types of molecular species with highly overlapping absorption spectra (sample heterogeneity).

Relaxation

VibrationPure electronic

energy

LUMO

Inte

nsity

HOMO

S1

S0

Fluorescence

1/energy

Vibronicband

Vibrationalband

FluorescenceAbsorption

Electronic

Rotation

Figure 5.5 The complex scheme of energy levels of polyatomic molecule. On the right: the schematic presentation of absorption and fluorescence spectra of polyatomic molecules.

Rupan_c05.indd 72 8/29/2012 5:37:49 PM


Therefore, absorption spectra of complex compounds can be as broad as several dozens of

nanometres and asymmetric due to a contribution of vibrations to their higher energy state

that is located on the left from the absorption maximum (see Figure 5.5 ). Absorption spectra

can therefore be a rich source of information about the concentration, identity of a molecule

or, in case of multicomponent system, number and types of compounds in a sample, their

immediate environment, interactions, and so on.

5.3 Fate of Absorbed Light Energy

After approximately 1 fs a number of events associated with the excited moving electron

can take place. First of all, according to the Frank-Condon principle, during the time of

electron movement into the excited state nuclei of molecule do not move, they are too inert

to follow the changes in orbital occupation by the excited electron. Following absorption,

nuclei will rearrange to adapt or rather to relax to accommodate the excitation. This

rearrangement is relatively fast, within 1 ps time scale. The reason for such rearrangement

is the term reorganization energy (Em) which appears as a result of the existence of a

coupling between electron and nuclear motions. What is the cause of the reorganization?

Well, excitation leads to changes in the spatial distribution of the electron. Since the

electrons have moved the nuclear arrangement of the ground state will now be subject to an

electrostatic force that was absent in the ground state and will rearrange accordingly.

Hence, nuclei ‘feel’ changes in electron energy as a result of absorption of light. Within

1 ps, nuclear coordinates will change in the excited state and a fraction of excited energy

will be lost nonradiatively as heat, whilst the electron is still in the excited, S1 state. This

energy loss is inevitable but relatively small. Following this relaxation a molecule can live

a few nanoseconds in the excited state with the excited electron occupying the LUMO.

Figure 5.5 shows absorption and fluorescence spectra of chlorophyll. The point of energy

where they overlap corresponds to the pure electron transition energy, the energy that is not

affected by nuclear motion. This is known as Frank-Condon or vertical transition. The

energy difference between maximum of absorption and maximum of emission is called

Stokes shift and is broadly equal to the amount of energy lost during nuclear reorganization.

For chlorophyll it is normally within 10 nm or ~200 cm −1 . The latter is in units that are

directly linear to the energy scale and called wavenumbers, ν = 1/ λ , calculated in cm −1 rather

than nm −1 . For a pigment absorbing at 660 nm, the wavenumber is 10 7 /660 = 15151.52 cm −1 .

If the fluorescence of this pigment is centred at 670 nm, then the Stokes shift will be:

10 7 /660 − 10 7 /670 = 226.15 cm −1 .

The lifetime of the excited state varies among different types of molecules. First of all it

is determined by t 0 , radiative lifetime: the fundamental molecular constant that can be

expressed as:

( )

τυ ε υ υ

=∫0 2 2

0

1,

2900n d

(5.5)

Rupan_c05.indd 73 8/29/2012 5:37:50 PM


where ∫ e ( u ) d u is the integral over the molar extinction coefficient, u 0 is the frequency at

point of maximum intensity, n is the refractive index of the medium into which the light is

being emitted. Hence the radiative lifetime depends upon the molar extinction. Molar

extinction is determined by absorption cross-section, s , that is proportional to the oscilla-

tor strength, f . The latter depends upon the length of the transition dipole moment, as was

mentioned previously. These relations mean that the time the electron can potentially live

in the excited state is determined by the probability of transition of this electron into excited

state upon interaction with light. Parker ( 1968 ) calculated that molecules with a large

molar extinction of around 10 5 should possess the radiative lifetime of a few nanoseconds

that is more than a million times longer than the time required for electron to occupy the

excited state, S 1 . Clearly, absorption of light is a good means of temporarily storing energy.

Reemission of absorbed light, fluorescence is an obvious fate of absorbed energy, when

the excited electron returns to the ground state, losing its energy in the form of an emitted

photon: radiative energy dissipation . However, in reality this channel is often not a main

channel of de-excitation. Figure 5.6 shows four more alternative fates of electronic excita-

tion energy dissipation. Apart from fluorescence, internal conversion can take place, when

excited electron returns into the ground state losing the excitation energy via molecular

vibrations resulting in production of heat. Whilst the fluorescence rate constant, 1/ τ o for

chlorophyll is 0.06−0.07 ns −1 , the internal conversion rate, k IC

, is almost two times higher.

In the system with this internal conversion rate fluorescence lifetime will be not 15 ns ( t 0 ),

Fates of excitation energy

1 2Fluorescence

0.07 ns–1 0.14 ns–1 0.01 ns–1 20 ns–1 1–100 ns–1

Internalconversion

Tripletformation

5

4

1

S0

S1

2

3

T1

Photochemistry Energytransfer

KTKPKSTKICKf

3 4 5

Figure 5.6 Fates of excited energy in a molecule.

Rupan_c05.indd 74 8/29/2012 5:37:51 PM


but ( )1 / 4.8 nsf f ICk kτ = + = . This means that internal conversion shortens the excited

state lifetime and therefore the time light energy is being stored and is available for photo-

synthesis.

Another fate of excitation energy is intersystem crossing ( k ST

) that leads to the formation

of a triplet excited state. This takes place when the electron in the LUMO changes its

‘spin’. Therefore the remaining electron in the HOMO and the excited electron in the

LUMO have the same spin polarization, giving the whole molecule a net spin. Absorption

into a singlet state does not change the spin orbital momentum. Decreased coupling of the

excited electron with the rest of electrons, certain environmental factors, like presence of

strong fields, and so on can cause the change of spin leading to the transition of molecule

into the triplet state. The electron energy in the T 1 state is less than in the S

1 state. For chlo-

rophyll the event is less likely than the fluorescence with the rate constant of ~0.01 ns −1 and

a yield of a few percent. However, the triplet excited state can leave much longer than the

singlet one (hundreds of microseconds) with a transition to the ground state proceeding via

a slow radiative process known as phosphorescence.

The most interesting fate of energy from the photosynthesis point of view is photochem-

istry, light-triggered chemical reactions. This process occurs when excited electron leaves

the molecule from S 1 state to be donated to another molecule. The molecule that loses an

electron can be re-reduced within ~10 ms and be ready for absorption of another photon.

Therefore, there can take place as many as 100 photosynthetic charge separation cycles per

second. Such a process occurs in the reaction centres of photosystems I and II starting the

chain of electron transport events that further stabilize the energy of Sun, making the light

harvesting process less and less reversible and converting this energy into an energy of

stable chemical bonds of the universal biological fuel ATP and reducing agent NADPH

(see previous chapters). The rate of photochemical reactions in the photosynthetic reaction

centre chlorophylls can be as high as 20 ns −1 with a potential quantum yield of 20/(20 + 0

.07 + 0.14 + 0.01) = 99 %. This means that the initial energy stabilization stage of the light

phase of photosynthesis is extremely efficient. Energy of almost every photon can poten-

tially be converted into the energy of moving electrons.

5.4 The Need for the Photosynthetic Antenna and the Fifth Fate of Excitation Energy

Photosynthesis evolved in anaerobic atmosphere where organisms lived in shade to avoid

the damage by UV radiation. Under these conditions, when the light intensity can be as low

as 50 or even less μ M m −2 s −1 or (6.02 × 10 17 . 50) photons, one quantum is absorbed by

chlorophyll only per 1–2 seconds. These figures can be obtained using the relationship

between the absorption cross-section, s , and e − molar extinction:

( )310 10,

A

ln

N

εσ =

(5.6)

Rupan_c05.indd 75 8/29/2012 5:37:51 PM


where N A is an Avogadro number. Absorption cross-section given in Å 2 and is for the e in

maximum chlorophyll absorption (~10 5 M −1 cm −1 ) ~ 4 Å 2 . Number of red photon absorption

acts per second will be equal to ( light intensity × s ) . Converting the light intensity from μ M

m −2 s −1 into μ M Ǻ −2 s −1 we obtain (0.3 × 4) = 1.2 photons per chlorophyll per second.

To utilize reaction centres more effectively the cheapest strategy in biosynthetic terms is

to synthesize light-harvesting pigment-proteins that increase area of cross-section of absorp-

tion and feed more quanta per second into the reaction centres, which can possess a turnover

rate of 100 per second (see previously). This means that there is a need to increase the rate of

energy delivery to the reaction centre by the order of two or even more orders of magnitude.

The first evidence of the existence of the photosynthetic antenna was obtained by experi-

ments of Emerson and Arnold some 80 years ago using fast (10 μs) flashes of light in order

to obtain one turnover (oxidation-reduction cycle, described previously) per flash (Emerson

and Arnold, 1932 ). The rate of turnover was found to reach 100 per second. They also found

that approximately 2500 chlorophylls are able to cause the evolution of one oxygen mole-

cule. Despite this, the efficiency of the light use corresponded to one molecule of oxygen

evolved per eight light quanta that were absorbed. According to the law of photobiology, one

quantum can drive one photochemical act. Therefore, one quantum absorbed in the popula-

tion of ~300 chlorophylls in the photosynthetic membrane causes one redox reaction (turno-

ver) in the reaction centre. This observation led to an unexpected conclusion that one reaction

centre of photosynthesis must be served somehow by ~300 molecules of chlorophyll. The

conclusions of Emerson and Arnold ( 1932 ) have been confirmed in several independent

experiments dealing with the estimation of effective cross-sections of the photosynthetic

reaction centres as well as the analysis of the ratio between various electron transport chain

components (cytochromes, reaction centre chlorophylls, etc.) and the total chlorophyll in the

membrane. There were simply too many chlorophyll molecules that seem to have no photo-

chemical activity at all. These observations led to emergence of the concept of the photosyn-

thetic unit, where majority of pigments are ‘serving’ one reaction centre with the absorbed

energy of light. But how can this process occur? What events should take place among the

pigments? So far we have considered the fates of excitation energy concerning one molecule.

However, the photosynthetic membranes are densely populated with proteins that carry pig-

ments at high concentration, meaning that they can potentially interact with each other.

Indeed, close positioning of pigments within each other in the photosynthetic membrane

is one of the major conditions at which these pigments can exchange excitation energy of

electrons. This process is called energy transfer and is the fifth channel of excited state

energy dissipation in chlorophyll. This process is called inductive resonance and was quan-

titatively described in Förster’s theory (1948). The simple resonance energy transfer

scheme is described by Equation 5.7:

+ = +* *,D A D A (5.7)

where D * and D is a molecule that gives energy ( D , donor) in excited and ground states,

respectively; A * and A is a molecule that receives energy ( A , acceptor) in excited and

Rupan_c05.indd 76 8/29/2012 5:37:52 PM


ground states, respectively. The event of energy transfer is similar to the phenomenon of

electromagnetic induction that can be observed in the macroworld. Molecules behave as

electromagnetic field oscillators that could emit, absorb, or indeed, give the electromag-

netic energy to each other. Förster ( 1948 ) derived the formula that enabled him to obtain a

rate constant for this energy transfer, k T :

6

0 ,T f

Rk k

R

⎛ ⎞= ⎜ ⎟⎝ ⎠

(5.8)

where k f is the fluorescence rate constant of a donor (D), R 0 is a distance between donor and

acceptor molecules at which the efficiency of energy transfer is 50% of the maximum, R is

the actual distance between donor and acceptor molecules. Remarkably, the Förster transfer

rate is steeply dependent on the distance between pigments, since it is proportional to the

sixth degree! R 0 was found to depend upon a few key parameters: (a) mutual orientation of

molecules ( k ), more accurately the direction of electronic transitions between states (transi-

tion dipole moments); (b) fluorescence yield of the donor ( f D ); (c) the refraction index of the

medium ( n ); and (d) the overlap between the fluorescence spectrum of donor ( f D ) and absorp-

tion spectrum of acceptor ( e A ) expressed via an energy overlap integral ( J = ∫ f

D e

A u − 4 d u) :

( ) 2

6 4

0 6 4

9000 10~ ,

128

DD A

lnR f d

n N

κ ϕε υ υ

π−∫

(5.9)

where u is the wavenumber, and N is Avogadro’s number. The orientation factor k = 2/3 in a

situation where pigments are randomly oriented, but can range from 0–4. For randomly ori-

ented chlorophyll molecules R 0 was reported to be about 80 Ǻ. Taking into account the size

of a chlorophyll molecule (~8 Ǻ) this is a very significant distance. If R ~ 25 Å , the k T will be

approximately 100 ns −1 , this is comparable with the photochemical rate constant. Therefore,

the energy transfer between pigments can be extremely efficient, provided they are oriented

and positioned in space appropriately and their energy overlap integral is reasonably large.

This means the pigments should be spectrally not too different, having similar excited state

energies. The other implication of Förster theory is that the rates of forward and backward

energy transfer from the acceptor to the donor can be similar, or can indeed be very different.

The latter case will take place when the excited state energy of acceptor is reasonably smaller

than that of a donor. In this case the D-A energy transfer rate will be faster than A-D energy

transfer rate. Figure 5.7 illustrates this idea showing absorption and fluorescence spectra of

the pair of pigments. It is clear that in this case the forward energy transfer rate should be at

least an order of magnitude higher than the back transfer rate. Here, the excited state of

acceptor is lower than that of a donor. When these levels are close, then the mentioned rate

constants become less different, since the respective overlap integrals should be less differ-

ent. At some point of shift between the excited state energies of donor and acceptor the

overlap integral of back energy transfer will become zero and this transfer will be impossible,

Rupan_c05.indd 77 8/29/2012 5:37:52 PM


only forward transfer will take place. In reality it may not be a likely event but ideally this is

a case of ‘trapping’ or localization of excitation energy on a single molecule.

It is important to mention that in a solution of pigments, when their average distances are

about 100 Ǻ their molar concentration should be about 2 mM. This concentration is already

high enough to observe reasonably strong concentration quenching described by Beddard

and Porter ( 1976 ). This energy de-excitation channel was attributed to molecular collisions

with each other and transient or sometimes stable formation of quenching pigment associa-

tions. Hence, effective energy transfer among 300 chlorophylls of the photosynthetic unit

derived from experiments of Emerson and Arnold was in danger of being undermined by

efficient energy dissipation due to the presence of concentration quenching. Indeed, as was

described earlier in this chapter, the molar cross-section of chlorophyll is small, 4 Ǻ 2 , and

the frequency of photon absorption at low light intensity is only about 1 per second. In this

situation, photoinduced charge separation in photosynthesis is far from being optimum,

since reaction centre turnover rate is ~100 per second, a hundred times faster than photon

absorption by one chlorophyll molecule. Hence a requirement for the action of many more

pigments emerges, as was indeed proved in experiments of Emerson and Arnold ( 1932 ).

This collection of photochemically inactive chlorophylls that feed the reaction centre with

excitation energy is called the photosynthetic antenna or light harvesting antenna .

Schematically the photosynthetic antenna is often shown as a funnel (Figure 5.8 ) channel-

ling energy of absorbed light in the form of electronic excitation within its pigments to the

reaction centre pigments that are photochemically active, able to donate electrons into the

electron transport chain. Antenna pigments must be organized efficiently in order not to

lose too much energy into various dissipative channels that were described earlier. The only

channel that is important is the energy transfer between antenna pigments, its efficiency

and directionality, so that the excitation could find its way relatively quickly into the reac-

tion centre. But how can those antenna pigments avoid concentration quenching if they are

Df

JAD

JDA

De Ae Af

Figure 5.7 Relationship between Förster spectral overlaps in cases of forward (JAD) and backward (JDA) energy transfer between the pair of donor (D) and acceptor (A) pigments. e and f indicate absorption and fluorescence spectra respectively.

Rupan_c05.indd 78 8/29/2012 5:37:53 PM


at high concentration in the photosynthetic membrane? Some decades after Emerson and

Arnold’s discovery (1932), biochemical studies revealed a number of pigment protein com-

plexes that build the antenna of photosystems (see Chapter 3). Association of pigments

with protein was certainly a solution. The last paragraph of this chapter, as well as Chapter

6, will explain this in detail and give examples.

The requirement for pigment interactions in the antenna is an obligatory feature or prop-

erty. Förster resonance energy transfer occurs in a condition when interpigment dipole-

dipole interaction energy is much smaller than the energy of their interaction with

environment (solvent). Normally at this condition vibrational relaxation of the excited mol-

ecule (see Figure 5.5 ) takes place before energy transfer can occur. However, it was

observed in solids or molecular aggregates of pigments, where pigment molecules can be

positioned much closer to each other and in particular regular orientations, interpigment

interaction energy can prevail over the energy of their interaction with environment.

Davydov ( 1964 ) formulated a theory of molecular excitons in solids that could explain a

number of important spectral phenomena that occur among chlorophylls in the photosyn-

thetic membrane. At certain intermolecular distances pigments can become excitonically

coupled. The energy of this coupling is described by the following equation:

1 2

12 3

12

,JR

μ μ=

(5.10)

where | μ 1 | and | μ

2 | are the magnitudes of the transition dipole moments of molecule 1 and 2

and | R 12

| is the intermolecular distance (measured as the separation of the centres of mass of

Heat

Fluorescence

AntennaTriplet

Photosynthesis

RC

Figure 5.8 The light harvesting antenna concept: the funnel of absorbed light energy for the photosynthetic reaction centre.

Rupan_c05.indd 79 8/29/2012 5:37:53 PM


the two molecules). In case of particular orientation of the transition dipole moments of the

two pigments splitting of the S 1 energy level (Davydov’s splitting) takes place, even if the

absorption spectra of the both pigments are identical. The extent of splitting is determined

by J 12 . The mutual dipole moment orientation defines the resulting spectrum appearance or

relative amplitudes of splitting components, one of which is of lower and the other of higher

energy then the energy of the monomeric pigment. Such an excitonically- coupled associa-

tion of molecules behaves as a single excitation holding unit. The exciton by definition is

delocalized across two or more pigments or, indeed, pigment domains. This excitonic state

persists until interactions of the component molecules with their respective environments

destroy the coherence that exists between molecules involved in the state, leading to emer-

gence of localized excitonic states or excitations of only one molecule that can be spread to

other pigments via the inductive resonance mechanism to other excitonically coupled

domains triggering the complex energy migration process. The delocalized excitonic states

occur mainly in solids, where pigments are well-organized and the environmental effects

leading to the loss of coherence of excited states are minimized. In biological systems such

as the photosynthetic membrane where pigment environment does not correspond to either

the liquid or solid phase medium, the coherent delocalized excitonic states are rare or short-

lived. They are frequently broken into localized excitons that migrate among pigment

domains or pigments, often by the inductive resonance process described earlier. Therefore,

the biological ‘soft’ matter of the photosynthetic membrane provides apparently an entropi-

cally-optimized environment where pigments in the photosynthetic unit are concentrated but

not entirely free to diffuse to encounter unwanted concentration quenching. They are not

entirely fixed either, to provide flexible excitation energy delocalization, migration and

delivery to the reaction centre. Compromise between excitonic states and the possibility of

the occurrence of highly unidirectional Förster energy transfer emerges as one of the key

features of photosynthetic unit organization. This compromise was recently addressed by

application of Redfield approaches to theoretical modelling of rather concentrated and het-

erogeneous pigment systems. Indeed, pigment organization in light harvesting antenna is not

as perfect as in a crystalline solid state. There are clusters of closely interacting pigments

separated from one another by larger distances. Therefore, the energy migration modelling

is more complex and includes excitonic as well as inductive resonance transfer features. In

addition, the protein environment and high pigment concentration are likely to modulate

internal conversion dissipative channel, which currently is very difficult to experimentally

assess and model.

As the distance between pigments becomes very short so that their electronic wave-

functions start to overlap with emergence of shared molecular orbitals promoting the

likelihood of yet another mechanism, Dexter electron exchange excitation transfer. This

transfer is a process that two molecules bilaterally exchange their electrons (Figure 5.9 ).

Indeed, whilst during inductive resonance, there is no substance transfer; Dexter transfer

requires the movement of electrons between two interacting molecules. Hence, this pro-

cess can be qualified as much localized electron transfer with zero-net charge transferred

within the molecular associate. It is unlikely to take place among large numbers of mol-

ecules in the photosynthetic unit of chlorophylls but rather occurs in specific minor cases

Rupan_c05.indd 80 8/29/2012 5:37:54 PM


that can be functionally very important, such as photoprotective excess energy dissipation

that will be described in Chapter 9. Unlike the six-power dependence of Förster energy

transfer rate, the rate of the Dexter mechanism decays exponentially when the distance

between pigments increases:

−2

~ ,DAR

LTk Je

(5.11)

where J is the energy overlap integral, as in Equation 5.9 and L is the sum of van der Waals

radii of the two molecules. This process occurs at faster rates than inductive resonance

energy transfer and is particularly significant for certain pigment-pigment associations and

when the excited state lifetime is very short (<1 ps) by nature of a pigment (see the later

description of xanthophylls energetics). The short excited state lifetime as well as very

small or zero value of transition dipole moment makes all other mechanisms of energy

transfer described previously rather unlikely (see Equations 5.9 and 5.10). Dexter transfer

does not depend upon the transition dipole moment and therefore can be a unique if not the

only way to transfer excitation between specific types of molecules, like carotenoids and

chlorophylls, positioned within van der Waals distance and closely interacting with each

other (see for example, Figure 3.5).

5.5 Photosynthetic Antenna Pigments

As a result of evolution nature has selected a relatively small group of pigments to serve in

the photosynthetic antenna: chlorophylls, phycobilins and carotenoids. Only very few of

them serve as photochemically active reaction centre pigments. The latter are chlorophyll,

in plants and algae, and bacteriochlorophyll, in photosynthetic bacteria. The antenna

pigments are more versatile. The reason for it will be discussed later in this chapter. The

photosynthetic antenna pigments of plants include chlorophylls a and b and several types

of oxygenated carotenoids, xanthophylls.

Figure 5.9 Schemes of Förster (inductive resonance) and Dexter (electron exchange) mechanisms of energy transfer.

Rupan_c05.indd 81 8/29/2012 5:37:54 PM


5.5.1 Chlorophylls

Chlorophylls are global pigments. Figure 5.10 shows a NASA satellite image of chlorophyll

a redistribution in the Biosphere made by SeaWiFS (sea-viewing wide field-of-view sen-

sor). The principle of this device is based upon reflection/colour spectral measurements

and enables to quantify chlorophyll concentration. Enormous areas of land and oceans

contain photosynthetic organisms that carry chlorophyll a giving them characteristic col-

our. The highest chlorophyll concentration was detected in polar waters full of microscopic

algae, like diatoms. In addition, equatorial waters and waters around the shores also contain

plenty of photosynthetic organisms. On land, the most intense chlorophyll signal was

detected from the areas of tropical forests and the vast lands of North American and

Siberian forests.

Why did chlorophyll become a major photosynthetic pigment? One of the reasons is

that, as was mentioned earlier, it possesses very high molar extinction of about 10 5 and

large absorption cross-section of 4Ǻ 2 . This makes it a very effective light capturer. The

radiative lifetime is reasonably long, ~15 ns, and the excited state lifetime in dilute solu-

tions is about 5 ns: nearly three orders of magnitude longer than the rate of photochemical

reaction or energy transfer, meaning that it is an effective transient energy storage pigment.

In addition, chlorophylls possess electronic first excited state (S 1 ) energy of around 1.8 eV

or ~42 kcal mol −1 . This is equivalent to the energy of nearly six molecules of ATP. This

means that the energy initially stored in chlorophyll in the excited state is large enough to

drive the process of the light phase of photosynthesis that ends with accumulation of ATP.

Structurally, chlorophylls are also suitable molecules since, as was discussed in Chapter 3,

they are amphiphilic, lipid-like molecules, natural to the membrane or more accurately,

membrane-bound protein environment.

The part of a chlorophyll molecule that is involved in photon absorption is a macrocycle:

a derivative of a tetrapyrrole, porphyrin ring that contains a coordinated magnesium atom

in its centre (Figure 5.11 ). In chlorophyll the ring is modified by oxidation to make a

chlorin ring. In addition a fifth, unconjugated ring is added by derivation from propionic

acid (C13) adjacent to the ring III. Chlorophyll’s a C7 atom is methylated, whereas in chlo-

rophyll b the group is replaced by a formyl group (Figure 5.12 ). Chlorophyll rings are

normally labelled with Roman numerals I–V clockwise. The hydrophobic phytol tail is

attached to the fourth ring. Phytol is very flexible and therefore can adopt various positions

and orientation relative to the chlorin ring. The plane of the chlorophyll is typically said to

define a local xy-plane where the x- and y-axes are defined so that they pass through rings

II–IV and I–III respectively (Figure 5.12 ). The chlorin ring of chlorophyll is the actual

chromophore , the light-absorbing part of the molecule. This chromophore is defined by 24

π -electrons which are delocalized across the conjugation path defined by the alternating

single/double carbon-carbon bonds which make up rings I-III. Laying above these occu-

pied π -orbitals is an identical number of unoccupied π -orbitals. Transitions between these

occupied and unoccupied orbitals, particularly between those near the band gap between

the Highest Occupied Molecular Orbital (HOMO) and the Lowest Unoccupied Molecular

Orbital (LUMO), have energies comparable to those of visible light. For illustrative

Rupan_c05.indd 82 8/29/2012 5:37:54 PM

Figu

re 5

.10

Glo

bal r

edis

trib

utio

n of

chl

orop

hyll

a . R

eprin

ted

with

per

mis

sion

from

http

://o

cean

colo

r.gsf

c.na

sa.

gov/

SeaW

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. © S

eaW

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Rupan_c05.indd 83 8/29/2012 5:37:54 PM


purposes schematic diagrams of the HOMO and LUMO of chlorin are shown in Figure 5.11 .

These orbital functions (or more correctly the square of these functions) represent

probability distributions, indicating the likelihood that the electron will be located at a

certain point around the ring.

The absorption spectrum of chlorophylls has two regions in the visible light spectrum.

One is located in the red part and corresponds to the absorption into the first excited state,

S 1 (Figure 5.13 ). The other region is in the blue site of the visible light spectrum and cor-

responds to the absorption into the second excited state, S 2 . It is often called the Soret

region or Soret band. The green colour of chlorophyll is due to reflection of green light that

is not absorbed by the pigment. There are two absorption peeks per each of excited state

regions, red and Soret, of chlorophyll reflecting the fact that the transition dipole moments

Chlorin

HOMO LUMO

Porphyrin

3

2

20

19

1817 15

14

13

12

11

10

9

8

7

65

NH

I II

IV III

HNN

N

4

16

1

Chlorin

3

2

20

19

1817 15

14

13

12

11

10

9

87

65

NH

I II

IV III

HNN

N

4

16

1

Figure 5.11 Structures of porphyrin and chlorin rings ( top ). The delocalized p -electron path is highlighted by dots. Probability of one electron location in the chlorin ring on the highest occupied molecular orbital (ground state) and lowest unoccupied molecular orbital (excited state) (bottom). (Courtesy of Christopher Duffy.)

Rupan_c05.indd 84 8/29/2012 5:37:56 PM

Mg

N

Y

VO

O

O

132 carbonyl

131 carbonyl

173 carbonyl

Polar groups1

23

Hydrophobictail

3

2

1

O

H

Chlorophyll-aH3C

* *

*

Chlorophyll-b O

H

2O

NN

N

X

I

II

III

IV

Figure 5.12 Structural formula of higher plant chlorophylls with indication of polar groups of chlorophyll involved in binding into the antenna protein environment. (Courtesy of Christopher Duffy.)

Abs

orpt

ion

Chl a

Chl b

QY (S1)

Chl b

Chl a

Qx

Wavelengh, nm

Soret(S2)

Phycocyanin

Fluorescence

S1

S2

S0

2 10–9S

10–12S

700600500400

Phycoerythrin

Carotenoids

Figure 5.13 Absorption spectra of chlorophyll a and b . Also schematically shown are regions of absorption of other photosynthetic pigments, carotenoids and phycobilins in order to demonstrate how light harvesting pigments of different photosynthetic organisms share the visible spectrum of Sun.

Rupan_c05.indd 85 8/29/2012 5:37:57 PM


lie along either the x or y molecular axis (Figure 5.13 ). The Q Y transition is much stronger

then Q X and is often referred to as the red absorption band. The vast majority of spectro-

scopic studies in the field of photosynthesis focus on this particular transition. The Soret

band possesses stronger extinction than the red band. However, the excited state is short-

lived, only ~1 ps (see Figure 5.13 ). This is due to a strong nonradiative relaxation via

vibrational transitions into the S 1 state. Naturally, during this process some energy is lost as

heat; however, since S 1 receives the energy from S

2 Soret absorption does contribute to light

harvesting. From S 1 energy can be dissipated via various channels described earlier in this

chapter. The fluorescence spectrum of chlorophyll is also displayed in Figure 5.13 . In part

it looks like a mirror image of red absorption. The Stokes shift is about 7 nm (~140 cm −1 ).

The vibronic satellite is normally ~15% of the main band maximum and is shifted into the

red by ~ 60 nm (~1200 cm −1 ).

Chlorophyll b is slightly less abundant than chlorophyll a and is present only in the light

harvesting antenna complexes. Reaction centre complexes and their core antenna compo-

nents (see Chapter 3) do not bind this pigment. The chlorophyll- b Soret band is red-shifted

relatively to that of chlorophyll a and the red band is blue-shifted. Therefore, simple struc-

tural modification (see Figure 5.12 ) creates a new pigment with a slightly different absorp-

tion profile. This means that chlorophyll b makes light harvesting antennae absorb light

over a broader spectral region than would be possible if the antennae bound chlorophyll a

alone. This is known as the spectral broadening of absorption cross-section. The funnel size

becomes larger (see Figure 5.8 ), hence more excitations per second can reach the reaction

centre (a cup in Figure 5.8 ). In addition, since the chlorophyll b Qy excited state energy is

higher than that of chlorophyll a the energy transfer between these pigments is highly uni-

directional (see Figure 5.7 ). Indeed, chlorophyll b to chlorophyll a transfer rate will be

much faster than the reverse transfer because the overlap integral in the b-a transfer rate

will be much higher than that corresponding to a-b transfer. This is another fundamental

advantage of having pigments with differing excited state energies. They can contribute

potentially to the directionality of the energy transfer in antenna analogous to the function

of gravity in the funnel displayed in Figure 5.8 .

The large chlorin ring has a potential to interact, via polar groups, with the protein envi-

ronment of antenna, as was already mentioned in Chapter 3. These groups are magnesium

atoms that can be involved in co-ordination interaction with certain polar aminoacids, like

histidine. In addition, three carbonyls, 13 1 C, 13 2 C (ether) (ring V) and 17 3 C (phytol

etherification site to ring IV) can potentially be involved in the hydrogen bonding. The

scheme in Figure 5.12 displays the principal potential sites of chlorophyll molecule anchor-

ing within the protein environment. The hydrophobic, flexible phytol tail will partition into

hydrophobic aminoacid domains or interact with phytols of other chlorophylls. Magnesium

could strongly anchor the centre of the chromophore to the protein and three carbonyls

could be involved in determining its orientation in order to adapt the direction of the transi-

tion dipole moments according to the energy transfer pathway requirements within an

antenna complex. Early biochemical studies on isolated antenna complexes revealed that it

was very difficult to remove chlorophyll from protein using only non-ionic detergents.

Ionic detergents, on the other hand would remove the pigment often leaving it free from

Rupan_c05.indd 86 8/29/2012 5:37:58 PM


magnesium in a form of phaeophytin. Hence, chlorophyll interacts reasonably strongly

with protein but not via covalent forces. Therefore, some conformational freedom is left to

enable a fine orientation of the pigment within its environment. Bound to antenna protein,

chlorophyll possesses a somewhat red-shifted spectrum in comparison to the pigment in

organic solvents like ethanol or acetone. Whilst in the latter state the maximum in the Q Y

absorption region occurs at about 662 nm, in the antenna environment it could be red-

shifted by up to 50 nm. Different microenvironments cause red-shifts of different ampli-

tudes, creating a range of chlorophyll spectral forms . Therefore, the absorption spectra of

antenna complexes are always red-shifted and broader that the spectra of isolated pigments

in solvents (see the next chapter). This broadening due to heterogeneity of the chlorophyll

environment is another beneficial feature of antenna organization, since it broadens the

spectral range of absorbed light, enhancing the cross-section and contributing to direction-

ality of the energy transfer.

5.5.2 Xanthophylls

Xanthophylls are one of the most abundant groups of pigments found in Nature. Every year

more than 100 million tonnes of them are being synthesized in the biosphere. Several hun-

dred molecular species of xanthophylls are currently known to exist. It is a functionally

versatile group that can act as powerful antioxidants, vitamin precursors, natural colorants

and odorants and therefore it is not surprizing that they are of global economic significance

contributing hundreds of millions of dollars to yearly trade.

Higher plant antenna xanthophylls, lutein, neoxanthin, violaxanthin and zeaxanthin are

among the most common xanthophylls on our planet (Figure 5.14 ). Neoxanthin is the most

polar of these xanthophylls, possesses a normally highly reactive allene group found rarely

in carotenoids. Neoxanthin is found to be almost exclusively in the 9- cis conformation in

nature. The significance of this structure is yet to be discovered. Violaxanthin, in contrast,

is a very symmetric molecule, containing two epoxy oxygen atoms on the end-ring groups.

Lutein is less oxygenated than violaxanthin and is asymmetric. It possesses two different

types of end-groups, β - and ε -rings, which differ by the position of the double bond within

the ring. Zeaxanthin, an isomer of lutein, is symmetrical. Zeaxanthin possess two β -ring

end-groups. Reversible de-epoxidation of violaxanthin into zeaxanthin occurs in the pho-

tosynthetic membrane, and is dependent on the light environment. As a result, an interme-

diate xanthophyll, antheraxanthin, which carries only one epoxy group, is transiently

formed. The variations in the end group structure and conformation are determined by the

carotenoid biosynthesis enzymes. These structural features are likely to determine localiza-

tion as well as functions of these xanthophylls in vivo .

Varying numbers of conjugated carbon double bonds in xanthophyll molecule affects

their delocalized excited state π -electron energy and therefore defines the colour. Indeed,

optical spectroscopic analysis of carotenoids is based on the fact that the 0-0 energy of the

first optically allowed transition is inversely correlated to the number of conjugated carbon

double bonds in the backbone of the molecule. Therefore, in theory, violaxanthin, lutein

and zeaxanthin, which have 9, 10 and 11 conjugated bonds should have all different

Rupan_c05.indd 87 8/29/2012 5:37:58 PM

Lute

in

Zea

xant

hin

0–

0

0–

1

0–

2

Vio

laxa

nthi

n

Neo

xant

hin

520

500

480

460

S2

Wav

elen

gth,

nm

440

420

400

0.0

0.1

0.2

0.3

0.4

0.5

0.7

0.8

0.6

0.9

1.0

1.1

1.2

1.3

Absorption

OH

HO

5'3'

1'

7'9'

11'

13'

15'

1513

119

71

53

OH

HO

HO

OO

5'3'

1'

7'9'

11'

13'

15'

1513

119

7

1 53

OH

OH

O

5'

5'

3'

3'

1'1'

7'

7'

9'

9'

11'

13'

15'

1513

11

Vio

laxa

nthi

n

Neo

xant

hin

OH

*

HO

Zea

xant

hin

Lute

in

9

11'

13'

15'

1513

119

7

15

3

15

7

3

Figu

re 5

.14

Xant

hoph

ylls

of h

ighe

r pl

ant p

hoto

synt

hesi

s. S

truc

tura

l for

mul

ae (

left )

and

abs

orpt

ion

spec

tra

in e

than

ol (

right

).

Rupan_c05.indd 88 8/29/2012 5:37:58 PM


absorption maxima positions of their second excited sate (Figure 5.14 ). The S 1 (or 2A

g ) is

symmetry forbidden with the transition dipole moment equal to zero. Hence the molar

extinction is zero. Neoxanthin has the same number of the conjugated double bonds as

violaxanthin, 9. However, the cis -conformation increases the energy of excited state lead-

ing to a slightly blue-shifted 0–0 transition. In addition to this shift, a characteristic cis -

band emerges at around 310–330 nm, which can also be used for distinguishing different

cis - isomers of the same carotenoid. Absorption maxima of lutein and zeaxanthin are fur-

ther red-shifted. One extra conjugation shifts the carotenoid absorption maximum to about

6 nm (~240 cm −1 ). Interestingly, unlike the chlorophyll absorption, spectrum of xantho-

phyll is much broader with a characteristic three band structure corresponding to signifi-

cant contribution of 0−1 and 0−2 vibrational transitions originating from a strong coupling

of electronic to vibrational transitions making the latter as strong the 0−0 transition

(Figure 5.14 ). The excited states of the xanthophylls are very short-lived in comparison to

chlorophyll, only a few hundred of femtoseconds, around four orders of magnitude shorter

then in chlorophyll. This makes xanthophylls potentially not very efficient light harvesting

molecules unless they engage in ultra-fast energy transfer with chlorophylls. This point

will be discussed later. As to the molar extinction, it is almost as high as for chlorophyll a

and the broad absorption spectrum as well as its complimentary coverage of part of a chlo-

rophyll Soret band absorption makes xanthophylls potentially good light harvesting pig-

ments (Figures 5.13 and 5.14 ).

Xanthophyll absorption is very sensitive to environment. Solvents with different polari-

ties and refractive indices significantly affect their optical properties. Because of the pro-

portionality of refractive index to the ability of a solvent molecule to interact with the

electric field of the solute, the excited state energy of a molecule is sensitive to it reflecting

in the effect on the absorption maxima positions. Figure 5.15 ( top ) shows three absorption

spectra of the same xanthophyll, lutein, dissolved in isopropanol, pyridine and carbon

disulfide. The solvent refractive indexes in this case were 1.38, 1.42 and 1.63 for the three

mentioned solvents, respectively. 0−0 transition maximum changes from 473 to 505 nm,

which is 32 nm or corresponds to approximately 1340 cm −1 or nearly 4 kcal mol −1 of energy.

This is an important example of how the molecular environment can dramatically affect the

excited state energy. Another spectral development can take place if the solvent mixture is

not able to maintain pigments in solute state. In this case formation of dimers and higher

aggregates of all plant xanthophylls is a very common phenomenon. Ethanol-water mix-

tures represent a good binary solvent system, which could not only yield xanthophyll

aggregates but also test hydrophobicity of these pigments using the solvent ratio at which

aggregation takes place (more detail will be provided in Chapter 8). Figure 5.15 (bottom)

displays three types of zeaxanthin absorption spectra: the pigment in solution, H- and

J-type aggregates. In H-type aggregate the transition dipole moments of the two xantho-

phylls molecules are in parallel orientation to each other. Since they are oriented along the

xanthophyll conjugated carbon backbone the molecular arrangement is called sometimes a

‘card-pack’. In J-type aggregate the transition dipole moments are arranged in a sequence,

called ‘head-to-tail’, as shown in Figure 5.15 . The H-type of aggregation causes a very

strong blue shift of the absorption spectrum duet to the domination of absorption of the

Rupan_c05.indd 89 8/29/2012 5:37:58 PM


high energy component of excitonic splitting (see the previous paragraph). J-type of aggre-

gation leads to the appearance of a red-shifted excitonic component. Here, the resulting

spectral variation in the measured mix of H- and J-type aggregates of zeaxanthin shown in

Figure 5.15 of more than 150 nm (~7500 cm −1 , 21 kcal mol −1 or just 3 ATP molecules), a

very significant difference, indeed. It is therefore important to bear in mind the dependency

of the carotenoid spectrum upon properties of the environment for in vivo analysis, which

is based on application of optical spectroscopies. This approach is often the only way to

study the composition, structure and biological functions of carotenoids. Spectral sensitivity

0.0400 420 440 460

Wavelength, nm

480 400

470 505∼1400 cm–1

380

H

Zeaxanthin

535

J

∼7500 cm–1

520 540

350 375 400 425 450

Wavelength, nm

500475 525 550 575 600

0.2

0.4

LuteinAbs

orpt

ion

Abs

orpt

ion

0.6

0.8

0.0

0.2

0.4

0.6

0.8

1.0

Figure 5.15 Absorption spectra of lutein in different solvents ( top ) and zeaxanthin is different states of aggregation ( bottom ).

Rupan_c05.indd 90 8/29/2012 5:37:58 PM


of xanthophylls to the medium could be a property to use for gaining vital information on

their binding sites and dynamics.

This presence of symmetric cyclic end-groups carrying oxygen atoms increases co-

ordination of the xanthophyll molecule in the membrane determining interaction patterns

with protein membrane-spanning helixes, as will be shown in the next chapter. Hydrogen

bonding plays an important role in the same way as the hydrogen bonding of chlorophyll

carbonyl groups to the protein environment. Xanthophylls bound to not only antenna but

other nonrelated proteins are likely to play important, yet currently not well-understood,

structural functions, similar to those of membrane lipids and beyond. Interestingly, lutein

and zeaxanthin have also been found in retina of humans and some primates. It is possible

that these carotenoids possess some universal photophysical properties essential for both,

photosynthesis and vision.

5.6 Variety and Classification of Photosynthetic Antennae

The photosynthetic antenna of different classes of organisms varies a lot in its pigment com-

position, concentration, number of protein subunits and their interaction with the reaction

centre. The reaction centres themselves are far more conservatively organized than antennae.

Here the crucial factor that led to the diversity of antenna systems is light environment.

Availability of light for different photosynthetic organisms, adaptations to very low light

environment or to specific spectral compositions of available light exerted a selection pres-

sure during the evolution of the early life on our planet. The first photosynthetic organisms

were prokaryotes, various types of bacteria that utilize light energy to drive their metabolic

processes. Their antenna systems vary considerably. Photosynthetic bacteria use different

types of bacteriochlorophyll as the major photosynthetic pigment in the reaction centre as

well as antenna. Xanthophylls are also present. The structure of one of them, rhodopsin glu-

coside, was already shown in Chapter 4. The S 1 absorption energy of bacteriochlorophyll is

localized in the infrared region of 800–900 nm. The structure of bacterial antenna system is

very different from the structure of antennae of higher plants. Some comparison of these will

be presented in the next chapter in order to understand evolutionary patterns in antenna func-

tion in general. Another class of photosynthetic organisms, blue-green algae or cyanobacteria

carry phycobilins as the major light harvesting pigments. They are open-chain tetrapyrroles.

Their absorption lies within the region where chlorophyll does not strongly absorb,

550–650 nm. Hence these organisms found their own share of the visible spectrum so that

they do not practically compete for light energy with photosynthetic bacteria or green algae.

In a similar manner to higher plants, the latter contain chlorophylls as well as various xantho-

phylls in their antenna: chlorophylls a , b , c and d as well as variety of different types of

xanthophylls with absorption extending up to 550 nm and more, with some also containing

phycobilins. The photosynthetic algae is the great example of chromatic antenna system

evolution that enabled their biodiversity and adaptations to particular wavelength regions of

sun light and co-exist with the other groups of photosynthetic organisms. Altogether their

various types of antenna cover then whole spectrum of sunlight (see Figure 5.13 ).

Rupan_c05.indd 91 8/29/2012 5:37:59 PM


Apart from different types of photosynthetic pigments, there exist different classes

of antenna design often showing no evolutionary connection that suggests that the

photosynthetic antenna emerged in several independent evolutionary ways. Thus, there

exist two major types of photosynthetic antenna: integral antenna, built of integral

membrane proteins and peripheral antenna that are not integral to the membrane but associ-

ated with it and reaction centre complexes via noncovalent (often electrostatic) interactions

(see Figure 5.16 ). Integral antenna can be of three types: fused, core or accessory antenna.

A fused antenna is integrated into the reaction centre, sharing the same polypeptide. A

typical example of a fused antenna is the photosystem I reaction centre complex that con-

tain two heavy polypeptides (see Chapter 3) that bind the photochemically active reaction

centre chlorophyll as well as about 100 chlorophyll a molecules serving only as light har-

vesting pigments. Core antenna complexes are intimately associated with reaction centres

but they do not share the same polypeptides. Typical examples are the core antennae of

photosystem II, the CP43 and CP47 complexes that bind only chlorophyll a and the LH1

complex of purple bacteria. Accessory antenna complexes are built of proteins that can be

arranged separately from the reaction centre complex and adopt various localizations rela-

tive to it. Typical examples are the major LHCII complex of photosystem II, the LHCI

complexes of photosystem I and the LH2 complex of purple bacteria. Finally, peripheral

antenna complexes associate with components of the reaction centre complexes that are

embedded in the photosynthetic membrane but do not themselves cross it. Typical repre-

sentatives are the phycobilisomes of cyanobacteria and red algae, the chlorosomes and

FMO complex of green bacteria and the peridinin-chlorophyll proteins of Dinoflagellates.

This classification is based on how autonomic the photosynthetic antenna is from the reac-

tion centre complex. It will become clear from next chapters that autonomicity of antenna

organization is important to enable prompt and efficient adaptation of the photosynthetic

membrane to the light environment.

Peripheral antennae

Antenna

RC Membrane

Integral antennae

RC + AntennaAntenna

AntennaRC

RC

CoreFused Accessory

Figure 5.16 Types of photosynthetic antenna.

Rupan_c05.indd 92 8/29/2012 5:37:59 PM


5.7 Principles of Light Harvesting: Summary

It is important now to summarize the design of the photosynthetic antenna based on

the analysis of its pigment composition, their spectral properties, their binding to the

protein, etc. The principles of light harvesting antenna design in the photosynthetic

membrane can be listed as:

1. The antenna utilizes pigment types with high molecular optical cross-section (large

chlorin ring-based molecules, the chlorophylls, and long linear polyenes, the carotenoids).

Both classes of molecule are archetypal examples of conjugated π -electron systems.

2. The antenna binds pigments that are relatively easily spectrally tuneable by their inter-

action with the environment or with each other. These pigments normally have rela-

tively broad absorption bands.

3. The antenna possesses an extremely high pigment concentration (often up to 1 M) while

simultaneously avoiding wasteful energy dissipation due to concentration quenching.

4. The antenna protein is of paramount importance to its function. It enables pigment

binding, defines their orientation within the environment, tunes their excitation

energies, determines the energetics and character of pigment-pigment interactions and

ultimately the overall efficiency of the antenna. Figure 5.17 displays the concept of the

antenna protein as ‘ a programmed solvent ’. It is genetically programmed to bind pig-

ments at certain sites, to interact with them and thereby tuning their spectral proper-

ties, hence modulating both the light harvesting and the adaptive properties of antenna.

5. The modular structure of the antenna enables its discrete organization, allowing for size

adjustment and repair.

6. The dynamic character of antenna organization allows for the lateral mobility and reor-

ganization which is essential for the adaptation of the photosynthetic membrane to the

Protein of antenna: “a programmed solvent” concept

Pigment binding

Primary structure determines:

Pigment tuning

Pigment orientation

Efficiency of pigments

•

•

•

•

MRKSATTKKVASSGSPWYGPDRVKYLGPFSGESPSYLTGEFPGDYGWDTAGLSADPETFSKNRELEVIHSRWAMLGALGCVFPELLSRNGVKFGEAVWFKAGSQIFSEGGLDYLGNPSLVHAQSILAIWATQVILMGAVEGYRIAGGPLGEVVDPLYPGGSFDPLGLADDPEAFAELKVKELKNGRLAMFSMFGFFVQAIVTGKGPLENLADHLADPVNNNAWSYATNFVPGK

Figure 5.17 The role of protein in the photosynthetic antenna.

Rupan_c05.indd 93 8/29/2012 5:38:00 PM


light environment. This ensures a ‘correct’ level of energy input into the photosystems

and an overall safe and efficient light harvesting process.

There exist three fundamental properties or parameters of the photosynthetic light harvesting antenna which determine its efficient function:

1. absorption cross-section : corresponds to the size (number of pigments and their

efficiency at feeding the reaction centre with excitation energy);

2. excitation energy lifetime : the time of energy storage within the antenna before it reaches

the reaction centres. This is an important parameter that depends on various pigment

associations and states that determine the overall excited state lifetime of antenna; and

3. energy migration efficiency and delivery to the reaction centre. This parameter is largely

determined by pigment positioning, mutual orientations and their spectral properties.

The absorption cross-section depends on the type and number of pigments, spectral redis-

tribution of extinction coefficients and the oscillator strengths of the electronic transitions

of the absorbing species. These are adjustable within long-term and even short-term light

adaptation processes and vary significantly among different types of photosynthetic organ-

isms. Spectral quality variations are of particular importance since they enable coexistence

and hence the biodiversity of photosynthetic organisms, particularly those living in low

light environments or aquatic ecosystems; purple, green and cyanobacteria, red, diatom and

green algae.

The time during which the antenna remains excited is the other important parameter. It is

generally proportional to the probability of energy delivery to the reaction centre and

largely depends on the environment surrounding the chlorophylls. The latter could influ-

ence chlorophyll conformation, directly interfere with the optical π -electron configuration

or remove excitations via energy transfer. Carotenoids, amino acids and lipids are potential

modulators of the antenna chlorophyll lifetime. The excitation lifetime can be monitored

by fluorescence lifetime or yield measurements, which reveal their remarkable dynamics,

reflecting the existence of control over the light harvesting process in plants.

The antenna protein functions as a tuner of pigment energies, determining the energy

transfer pathways within it. Figure 5.18 shows evidence for the energy transfer between

chlorophylls of isolated light harvesting complex II from plants (the major LHCII). It

displays two spectra. The dashed line spectrum is the fluorescence spectrum of LHCII

measured at 77 K in order to narrow the bands and increase spectral resolution. Fluorescence

is typically excited in the Soret band at around 435 nm. The spectrum shown in solid line is

a fluorescence excitation spectrum measured in the same experiment. The fluorescence is

detected at its maximum or, more often, at longer wavelength up to the vibronic satellite

region of 740 nm to avoid overlap with the excitation light that can be seen by the detector

as a result of scattering. The latter can be minimized by using transparent samples, special

sample compartment geometries and polarizers. The scheme of energy levels (Figure 5.18 ,

bottom panel) shows that different pigments, or indeed pigment forms, transfer energy

directionally down to the terminal emitter where it is being localized and emitted as

fluorescence. Remarkably, no fluorescence from chlorophyll b (which absorbs at 650 nm

Rupan_c05.indd 94 8/29/2012 5:38:01 PM


and is expected to emit at around 655 nm) is present in the fluorescence spectrum.

Fluorescence is neither observed from chlorophyll a forms absorbing at 660 nor 670 nm.

The maximum of LHCII fluorescence is centred at 680 nm and corresponds to the absorption

at around 676 nm or slightly longer with a small Stokes shift. Contributions to the excitation

fluorescence spectrum from chlorophyll b and xanthophylls are present, indicating that

these pigments transfer energy to the terminal emitter chlorophyll a . The excitation

fluorescence or action fluorescence spectroscopy was one of the first methods that

established the fact of energy transfer between antenna pigments connected in the funnel

fashion, so that energy from blue-shifted pigments is transferred downhill towards the red

pigments. This energy finally reaches the terminal emitter, the ‘collector’ of the energy

absorbed by the whole antenna. This setup therefore functions as a true energy funnel.

Provided the antenna is appropriately positioned in the vicinity of the reaction centre, the

Wavelength, nm

Fluorescencespectrum

4000.0

0.2

0.4

0.6

0.8

Flu

ores

cenc

e, e

l. Xanthophylls

Chl b

Chl b

660Excitation

flourescencespectrum

676 680

670

650

Chl a

Chl a

S2 S1

1.0

1.2

1.4

1.6

450

RC

ElectronRed

emitter

Terminalemitter

Light harvesting antennapigments

ΔE

500 550 600 650 700 750

Figure 5.18 Evidence for energy transfer in the major light harvesting complex of plants.

Rupan_c05.indd 95 8/29/2012 5:38:01 PM


energy that is collected by the terminal emitter chlorophyll can be transported to the reaction

centre for trapping and stabilization prior to eventual charge separation and electron transfer.

Careful analysis of the structure of excitation fluorescence and reversed transmission

spectra is a useful approach that can yield information about the energy transfer efficiency

from various pigments to the terminal emitter (see Chapter 4). A curve fitting technique

based on the mathematical deconvolution of multicomponent spectra into individual bell-

shaped components (most often Gaussian or Lorentzian functions) is used for this pur-

pose. It produces the areas below each component and compares them for two types of

spectra, excitation fluorescence and reversed transmission. This comparison, along with

simple calculations, yields the energy transfer coefficients. When LHCII is subjected to

high temperature treatment or incubation with a strong detergent that denatures the protein

and disconnects pigments the excitation fluorescence spectrum becomes much simpler,

narrower, showing only bands of mainly one chlorophyll a . The entire regions from

460–520 and from 630–650 nm will be missing since chlorophyll b and the xanthophylls

will be energetically uncoupled from chlorophyll a .

Protein as a key conductor of pigment properties ensures the efficient funnel-like energy

transfer properties of the light harvesting antenna. As a result, instead of trapping an amount

of energy equivalent to only one photon per second in a low light environment (see previ-

ously) the reaction centres receive 200–300 times that amount from the antenna. Hence, the

antenna is a key power enhancer of the photosynthetic apparatus and therefore an enhancer

of plant growth and productivity in general. The photosynthetic antenna is built of several

discrete pigment-protein complexes, organized in monomeric and/or oligomeric units

associated differently with the proteins carrying the reaction centre (see Chapter 3). This

arrangement allows gradual regulation of antenna size according to the environmental light

intensity and flexible repair of the reaction centre components in the case of their photoda-

mage. The differential association of the light harvesting antennae allows loosely associated

units to undergo diffusion in order to rearrange themselves within the membrane (see

Chapter 9 for more detail). Such rearrangements are normally of an adaptive nature, help-

ing photosystems to cope with fluctuations in environmental light quality and variations in

intensity in order to optimize and protect the functionality of the photosynthetic membrane,

specifically the delicately balanced charge separation processes in the reaction centres.

Therefore, the modular and dynamic character of natural antenna design on the nanoscale

ensures the durability and flexibility of the primary energy conversion processes of photo-

synthesis. This design enables a fundamentally important property of the antenna: regula-

tion and optimization of photosynthetic electron transport efficiency for the most economic

and safe performance of the light phase of photosynthesis.

5.8 Connecting Light Harvesting Antenna to the Photosystems: Red Energy Traps

Peripheral light harvesting antenna is connected to the photosystem complexes. This con-

nection is based on noncovalent interactions between antenna subunits and subunits of

reaction centre core complexes. In the case of PSII light harvesting antenna is attached to

Rupan_c05.indd 96 8/29/2012 5:38:01 PM


the core PSII containing CP43 and CP47 inner antenna complexes tightly interacting with

proteins carrying the reaction centre chlorophylls, D1 and D2. The reaction centre is almost

isoenergetic with the terminal emitter energy of LHCII. However, CP43 and CP47 com-

plexes possess terminal emitters slightly downshifted, around 683 and 690 nm respectively.

Both are red-shifted relatively to the absorption of P680, the reaction centre of chlorophyll.

In PSI, the redshift of the terminal antenna emitters does also take place relatively to the

RCI (P700) absorption, where the red-shifted chlorophyll forms absorb at around 710 nm.

The presence of red-absorbing emitters presumes the existence of the anti-Stokes energy

transfer, that is, from the lower energy level to the higher energy level of the reaction cen-

tres (Figure 5.18 ). At room temperature it is possible, since the energy difference is not

large (up to 10–15 nm) and fluorescence spectra of red-shifted donors overlap with broad

absorption spectra of reaction centre chlorophylls. The role of the red emitting chlorophyll

forms present in photosystems has been investigated for a number of years. One of the

major opinions suggests that they are essential in focusing excitation energy in the vicinity

of reaction centre ensuring its prompt localization around RC and constant balanced supply.

Figure 5.19 (top) shows low-temperature fluorescence spectra of PSII with dominating

fluorescence emission from CP43 (685 nm, known as F685) and CP47 (693 nm, known as

1.2

1.0 680685

693733

PSIPSIILHCII

660 680 700

Wavelength, nm

Flu

ores

cenc

e, r

el.

720 740 760 780

0.8

0.6

0.4

0.2

0.0

1.0

0.8

0.6

0.4

0.2

0.0650 700 750 800

PSI

PSlI

LHCIIFlu

ores

cenc

e, r

el.

Figure 5.19 Low temperature fluorescence spectra of photosystems and LHCII complex ( top ) and fluorescence spectrum of isolated chloroplasts ( bottom ).

Rupan_c05.indd 97 8/29/2012 5:38:01 PM


F695) complexes. The emission of LHCII antenna in this preparation is not clearly visible

since most of its energy is trapped by the red emitters at this temperature and therefore the

fluorescence is quenched and appears as a small shoulder, which is more pronounced in the

spectrum of whole chloroplasts (Figure 5.19 , bottom). Fluorescence spectrum of PSI is

even more red-shifted then that of PSII, peaking at 733 nm: a red emitter of this photosys-

tem, also known as F735. The fact that the chloroplast fluorescence spectrum is presented

by the both PSII and PSI bands suggests that these photosystems are separated in space by

the grana stacking as was discussed in Chapter 3.

Figure 5.20 shows electron microscopic pictures of stacked grana, where PSII is local-

ized and unstacked stroma thylakoids: a site for PSI localization. Magnesium cations are

required to keep grana stacked. Artificial removal of magnesium from the chloroplast incu-

bation medium leads to unstacking of grana, as shown in Figure 5.20 , with appearance of

long lamellae that contain mixed complexes of photosystems and LHCII. The first and

classical method of assessing whether in this state photosystems can interact with each

other is to measure excitation fluorescence spectra of the red emitting forms of PSI, since

they are the most red-shifted and can signal if PSII and LHCII are energetically coupled to

PSI. Figure 5.20 shows 77 K excitation fluorescence spectrum for F735 PSI band. At

stacked grana conditions, PSI excitation fluorescence spectrum is enriched almost with

chlorophylls belonging to this photosystem with low contribution from Chl b (650 nm) and

red-shifted Chl a maximum at 681 nm. In unstacked membranes, PSI cross-section (here,

read F735 cross-section) is much increased at the expense of Chl b and blue-shifted Chl a

(676 nm). The difference unstacked- minus -stacked spectrum is identical to excitation fluo-

rescence spectrum of PSII with attached LHCII antenna. This means that PSII with its light

Stacked

StackedUnstacked

Unstacked

PSI+PSII

PSI

PSII+LHCII

660 680 700620 640

1.0

0.8

0.6

0.4

0.2

0.0

Figure 5.20 Negative stain electron microscopy images of stacked ( top left ) and unstacked ( bottom left ) thylakoid membranes and corresponding excitation fluorescence spectra of photosystem I (F735) with the difference spectrum unstacked-minus-stacked, PSII + LHCII ( right ).

Rupan_c05.indd 98 8/29/2012 5:38:02 PM


harvesting antenna is channelling energy into PSI resulting in a strong increase in F735

emission of the PSI band. The 77 K fluorescence and excitation fluorescence techniques

have been used for many years in the photosynthesis research and gave first insights into

energy redistribution between the two photosystems and the impact of the structure of the

photosynthetic membrane on this process. Later, it will be shown how these approaches

were used in studies of composition and vital processes of light adaptation of the photosyn-

thetic membrane.

References

Beddard , G.S. and Porter , G. ( 1976 ) Concentration quenching in chlorophyll , Nature , 260 , 366 – 367 . Davydov , A.S. ( 1964 ) The theory of molecular excitons , Sov. Phys. Usp. , 7 , 145 – 178 . De Broglie , L. ( 1922 ) Rayonnement noir et quanta de lumière . Journal de Physique , 3 , 422 – 428 . De Broglie , L. ( 1923 ) Waves and quanta . Nature , 112 , 540 . Emerson , R. and Arnold , W. ( 1932 ) The photochemical reaction in photosynthesis . Journal of

General Physiology , 16 , 191 – 205 . Förster , T. ( 1948 ) Zwischenmolekulare Energiewanderung und Fluoreszenz , Ann. Phys. , 437 , 55 – 75 . Heisenberg , W. ( 1927 ) Über den anschulichen Inhalt der quantentheoretischen Kinematik und

Mechanik . Zeitschrift für Physik , 43 , 172 – 198 . Parker , C.A. ( 1968 ) Photoluminescence of Solutions: With Applications to Photochemistry and

Analytical Chemistry . Amsterdam, London, New York : Elsevier . Planck , M. ( 1900 ) Entropy and Temperature of Radiant Heat . Annalen der Physik , 1 , 719 – 737 . Schrödinger , E. ( 1925 ) An Undulatory Theory of the Mechanics of Atoms and Molecules . The

Physical Review , 28 , 1049 – 1070 . Schrödinger , E. ( 1926 ) Quantisierung als Eigenwertproblem . Annalen der Physik , 79 ( 361–376 ), 489 − 527 .

Bibliography

Atkins , P.W. ( 2000 ) Physical Chemistry . Oxford : Oxford University Press . Atkins , P.W. and de Paula J. ( 2006 ) Physical Chemistry for the Life Sciences . Oxford : Oxford

University Press . Blankenship , R. ( 2002 ) Molecular Mechanisms of Photosynthesis . London : Blackwell Science . Chang , R. ( 1981 ) Physical Chemistry with Applications to Biological Systems . New York : Macmillan

Publishing Company . Clayton , R.K. ( 1970 ) Light and Living Matter, Volume 1: The Physical Part . New York : McGraw-Hill . Clayton , R.K. ( 1970 ) Light and Living Matter, Volume 2: The Biological . New York : McGraw-Hill . Clayton , R.K. ( 1980 ) Photosynthesis. Physical Mechanisms and Chemical Patterns . Cambridge :

Cambridge University Press . Landrum , J. (ed.) ( 2009 ) Carotenoids: Physical, Chemical and Biological Functions and Properties .

Florida International University : CRC Press . Ruban , A.V. and Johnson , M.P. ( 2009 ) Dynamics of the Photosystems Cross-Section Associated with

the State Transitions in Higher Plants . Photosynthesis Research , 99 , 173 – 183 . Ruban , A.V. , Duffy , C.D.P. and Johnson , M.P. ( 2011 ) Natural light harvesting: principles and envi-

ronmental trends . Energy and Environmental Science , 4 , 1643 – 1650 . Serdyuk , I.N. , Zaccai , N.R. and Zaccai , J. ( 2007 ) Methods in Molecular Biophysics . Cambridge :

Cambridge University Press . van Amerongen , H. , Valkunas , L. and van Grondelle , R. ( 2000 ) Photosynthetic Excitons . Singapore :

World Scientific .

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‘ Study the past if you would define the future .’ Confucius

Research into the mechanisms of light harvesting has been going on for decades.

Methods of functional studies using various spectroscopies have been applied to reveal

various features of the process and understand its sheer efficiency and robustness. Along

with them, various isolation procedures for light harvesting complexes have been devel-

oped yielding essential material for spectroscopic studies that in most cases demanded

pure and simpler systems than those existing in vivo in the form of multi-subunit anten-

nae attached to the reaction centre complexes. In addition, newly emerging molecular

biology procedures of production of recombinant antenna proteins and their reconstitu-

tion with pigments enabled highly homogeneous individual pigment-protein complexes

to be obtained. Furthermore, some trials were successful in producing aminoacid point

mutants to probe pigment binding and assist the identification of chlorophyll a and b

spectral forms. However, the atomic resolution structure of higher plant antenna com-

plexes was always an ultimate goal for understanding the works of the photosynthetic

light harvesting. The path towards this goal was and remains not straight-forward,

exactly as the discoverer of the light harvesting complex II (LHCII), Philip Thornber,

predicted in 1975:

Towards the Atomic Resolution Structure of Light Harvesting

Antennae: On the Path of Discoveries

6

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… certain necessary studies will be more difficult, such as the development of an improved technique for isolating the existing and new chlorophyll-proteins in a stable form, as well as the determination of the three-dimensional organization of the chlorophylls within the protein framework .

Indeed, isolation of a membrane protein in intact state sounds almost contradictory, since

its natural environment is lipid bilayer in a membrane crowded with protein (see Chapter 3).

In addition, conditioning a membrane protein for crystallography imposes further artificial

environment factors likely to influence protein conformation. Finally, the ambiguity of this

structural approach is largely defined by the level of resolution, as it will be shown and

discussed in this chapter. It will become increasingly apparent that the structure and func-

tion of light harvesting antenna complex can only be understood when various approaches

and methods are combined together in a complimentary way with understanding of advan-

tages as well as limitations of each of them.

6.1 Discovery and Primary Characterization of the Higher Plant Antenna Complex

Postulation by Emerson and Arnold (1932, see Chapter 5) of the photosynthetic unit

concept in the 1930s and justification of the need for an antenna and the evolutionary

inevitability of its emergence were the first crucial advances in the studies of the phe-

nomenon of light harvesting. The next big step on this path came from biochemical

studies proposing the chlorophyll of the photosynthetic membrane is attached to protein

and hence is not free in the membrane. Indeed, the environment of chlorophyll in the

membrane always caught the attention of researchers. About 10 years after the discov-

ery of the photosynthetic unit and therefore the existence of a dominating amount of

photochemically-inactive chlorophyll, the first evidence emerged suggesting that chlo-

rophyll forms complexes with membrane protein. In the early 1940s Smith and Pickels

( 1941 ), who experimented with various surfactant-type agents, like bile salts (natural

fat-dispersing agents), detergents cholate, digitonin or sodium dodecyl sulfate (SDS)

aimed to dissolve (solubilize) chloroplast membranes. SDS treatment with subsequent

centrifugation enabled the observation of comigration of normally fairly light chloro-

phyll (~1 kDa) with a much heavier protein fraction. They also observed that anionic

detergent SDS produced a lot of free pigments. Nevertheless, the use of anionic deter-

gents became very popular in the studies of the photosynthetic membrane composition

manifesting in another discovery: the existence of several different chlorophyll-binding

proteins. This advance was made owing to the invention of polyacrylamide gel electro-

phoresis that enabled to separate two different pigmented protein fractions based on

their charge. These fractions possessed different chlorophyll a/b ratios. One had Chl a/b

ratio of about 5 and the other 1 : 2. Component II possessed much higher chlorophyll

amount than component I. However, the amount of free chlorophyll in these experi-

ments was far too high – 60–80%. Attempts have been made to reduce SDS/Chl ratio in

order to decrease the amount of free chlorophyll. Despite this, until the end of 1980s the

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Towards the Atomic Resolution Structure of Light Harvesting Antennae 103

uncertainty existed as to the origins of the free chlorophyll band in the preparations and

existence of the free pigment in vivo . The least mobile band, termed complex I, turned

out to be photosystem I: relatively stable complex made of large polypeptides (see

Chapter 3). Indeed, it turned out to be more resilient to the ionic detergents than com-

plex II, which possessed low Chl a/b ratio. The two complexes had not only different

chlorophyll but also carotenoid composition. Complex II was more enriched in lutein

and neoxanthin, whilst complex I was enriched in β −carotene. The origins of this com-

plex have been in the focus of intense research for some time. No photochemical activ-

ity has been detected on these preparations. Most interestingly, Thornber found that

whilst complex I was essential for survival of plants and algae, complex II, that com-

prised almost 50% of all membrane chlorophyll, was not. On the other hand, prepara-

tions of thylakoid membrane fragments using nonionic detergents digitonin and triton

X100 enriched in the complex II possessed photochemical activity. The fact that mutants

lacking this complex could survive autotrophically lead Thornber to suggest the follow-

ing in 1975:

This chlorophyll a/b-protein complex which accounts for such a large proportion of the chlorophyll and protein of the photosynthetic apparatus of higher plants is thus not essential for a plant to grow photosynthetically, and therefore a new name , light-harvesting chloro-

phyll protein, is proposed for the complex, which was formerly termed the photosystem II chlorophyll-protein .

Further, it was suggested that this LHCP (or more commonly referred to as LHCII) com-

plex acts as the photosynthetic antenna for photosystem II reaction centre, RCII. The fact

the complex II on the SDS gel did not possess the photochemical activity prompted to

propose that the method simply was not capable to identify whereabouts of the PSII reac-

tion centre as it did for PSI in the complex I. In addition, since the complex II was entirely

absent in the membranes of mutants lacking chlorophyll b , it was apparent that RCII com-

ponents could not be physically present in the complex II gel band.

In Chapter 3 we learned that the two photosystems have very different architecture. PSI

has its antenna integrated with the reaction centre on the same protein (see Chapter 5),

whereas PSII has a very large outer type of accessory antenna, LHCII complex that can

bind the majority of its pigments (average 80%), relatively minor core antenna and reaction

centre-carrying proteins (see Chapter 3). Therefore in the days of the use of a very strong

ionic detergent SDS, more stable complex, PSI, with integrated antenna could ‘survive’ the

treatment, whilst PSII was largely disintegrated losing its activity with a lot of pigments

washed away from it by the detergent. This event could also be explained by the potentially

weaker protein binding strength of chlorophyll a than chlorophyll b since the latter pos-

sesses additional polar group that could be involved in hydrogen bonding, a formyl car-

bonyl (see Chapter 5). Indeed, pigment composition of the free pigment fractions regardless

the isolation procedure tends to be enriched in chlorophyll a .

Discovery and isolation of light harvesting complex in higher plants and algae was the

major achievement in biochemical photosynthesis research that opened a path towards

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structural and functional characterization of the photosynthetic unit proposed by Emerson

and Arnold and specifically the organization of the light harvesting antenna. LHCII as an

outer antenna is by far the major light harvesting complex, dominating the membrane pro-

tein composition, hence it is not surprising it was discovered first and studied much better

than any other PSII and PSI antenna complexes (minor PSII light harvesting complexes,

inner antenna complexes, CP43 and CP47, LHCI complexes of PSI) that were isolated and

characterized later.

6.2 Development of Isolation Methods: Intactness, Purity and Quantity

Further biochemical work on the photosynthetic antenna focussed on the invention of vari-

ous techniques to analyse and prepare light harvesting complexes in significant amounts

and with a high level of intactness. However, this was not an easy task, since nobody knew

what was the native state of a light harvesting complex. How many pigments and pigment

types it should bind in vivo ? What oligomeric state of LHCII was the natural one?

Towards the end of 1970s Arntzen’s group made a major breakthrough in the isolation

in preparative amounts of reasonably pure LHCII complex (Armond et al ., 1977 ). This

work came from the study of the role of light LHCII complex in magnesium- and potas-

sium-dependent grana stacking (see Chapters 3 and 5). The process is vitally important in

the sharing of light energy or energy redistribution between PSII and PSI, otherwise known

as spillover. It was found that mutants lacking LHCII have a reduced ability for spillover.

In addition, for the high chlorophyll fluorescence state of PSII corresponding to its intact

state with LHCII assembled around the photosystem magnesium was absolutely required

(see Chapter 5, Figure 5.20). Therefore it was suggested that LHCII binds magnesium and

this triggers grana stacking and segregation of photosystems leading to a minimum spillo-

ver as well as promotes assembly of LHCII around the core PSII complex. At low cation

concentration, a certain extent of spillover takes place at which LHCII is less tightly bound

to PSII and shared by both, PSII and PSI, so that the excitation energy is more evenly

redistributed between them. Using this assumption Arntzen and coworkers treated isolated

thylakoid membranes with nonionic detergent Triton X-100 at conditions of low cation

concentrations at which grana were unstacked and LHCII was separated from PSII

(Armond et al ., 1977 ). The solubilized complex was separated on the sucrose gradient (see

Chapter 4) presenting itself as a broad highly fluorescent band. This band was removed

from the gradient, and magnesium and potassium salts were added to the sample. This

caused binding of magnesium and potassium cations to LHCII particles and resulting com-

pensation of negative charges that promoted their aggregation. Aggregated LHCII was

separated from the rest of the sample by centrifugation through the cushion of 0.5 M of

sucrose in the ultracentrifuge. Aggregates appeared in the pellet indicating the higher den-

sity of LHCII in the aggregated state. They could be resuspended back into solution by the

addition of detergent. Denaturing SDS gel electrophoresis of the prepared LHCII revealed

the presence of not one but at least two polypeptides of different intensity, suggesting the

heterogeneity of LHCII complex. This preparation yielded relatively large amounts of

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LHCII. The method immediately became one of the major procedures used in various

modifications for isolation of the complex for analytical and spectroscopic studies. It also

became a key method of LHCII preparation for the first crystallographic trials (see the fol-

lowing). As early as 1980 it was suggested by Ryrie and coworkers that the ability of

LHCII to form 2D lamellae in the low detergent solutions can be used for creation of

highly ordered arrays of the complex suitable for crystallography studies (Ryrie et al ., 1980 ). Indeed, LHCII preparations were relatively stable and the complex was easily self-

assembled into the 2D ordered arrays particularly when the detergent was removed gradu-

ally at certain temperature and buffer composition by dialysis. Kühlbrandt’s group in the

early 1980s was the first to obtain highly ordered 2D arrays of LHCII complexes induced

by cation precipitation at conditions of low Triton X100 concentration (Kühlbrandt, 1983).

Analytical separation of LHCII on polyacrylamide gels was further improved so that

instead of 50–60%, as in early experiments of Thornber, only 10% of pigments were

removed from protein and ended up in the free pigment zone (FP): the fastest running

band on the electrophoresis. It is often called ‘Anderson phoresis’ after Jan Anderson’s

papers published at the end of the 1970s/early 1980s (1980, 1982) (among some other

prominent groups such as Simpson’s: Bassi et al ., 1987; and Machold’s: Machold and

Meister, 1979 ) that showed this kind of separation. This method was based on the use of

ionic detergent, SDS of about 0.5% concentration only. This allowed separation of sev-

eral green bands from PSI. Three of them belonged to LHCII and were labelled LHCP 1 ,

LHCP 2 and LHCP 3 . The slowest running was LHCP 1 and the fastest, LHCP 3 . This was

the first demonstration of LHCII heterogeneity and it was not entirely clear whether it

was entirely due to the different molecular weight of its polypeptides or different oligo-

meric state or both. Other remarkable achievement was the presence of a small band

containing only chlorophyll a , that was called CPa: a found PSII reaction centre com-

plex. It possessed only 15% of the total membrane chlorophyll. This band was also pre-

sent in plants lacking LHCII and possessed fluorescence properties of the PSII reaction

centre complex. The three separated LHCII complexes had very similar pigment compo-

sitions and similar absorption and fluorescence spectra. Moreover, polypeptide analysis

revealed that they are built of very homologous polypeptides. This suggested that the

differences in mobility of the resolved LHCII complexes were likely to reflect different

states of their oligomerization. The electron crystallography approach used by Butler and

Kühlbrandt ( 1988 ) allowed the first projection map of negatively stained 2D arrays of

LHCII to be obtained. It was revealed that the crystallographic unit cell contained two

structural units, each possessing three-fold symmetry. Therefore, the authors proposed

that the LHCII of their preparation was an oligomer of three monomeric light harvesting

complexes that were built of the two types of polypeptides with 25 and 27 kDa molecular

weights. Some years later Kühlbrandt (1994) successfully used an analytical ultracen-

trifugation method to confirm that LHCII is a trimer. It was also suggested that each

monomeric unit regardless the molecular weight (25 or 27 kDa) possessed eight chloro-

phyll a s and seven chlorophyll b s.

For crystallography the modified LHCII preparation method from Arntzen was of

sufficient quality. The preparation was relatively stable but not essentially pure.

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However, the crystallization itself was another purification step since only two types of

polypeptides were found in the LHCII crystals. This meant that, although the initial

preparation could have been impure, only specific complexes were involved in the for-

mation of the good quality crystal. Therefore, the work of Kühlbrandt justly focussed

on obtaining the structure of LHCII trimer from such preparations. At the same time the

other path of LHCII antenna research was leading towards development of novel bio-

chemical analytical and preparative methods in order to obtain a separation of various

antenna complexes at the conditions of minimal loss of pigments. This would yield

useful information on the full composition of light harvesting antenna giving accurate

protein and pigment stoichiometries. Such information was essential for accurate func-

tional characterization of LHCII as well as to help improving of emerging structural

data. This work was carried out in the 1990s by several groups, most notably of Thornber

and Bassi (1997). The major idea was to use milder nonionic detergents than Triton

X100. In 1991, both groups produced work suggesting the existence of 4–5 types of

LHCII antenna complexes. Thornber called them LHCII a, b, c, d and e and Bassi

(1987), CP29, LHCII, CP26 and CP24, respectively. The fifth complex was missing in

the latter work. Hence, LHCII was named LHCIIb or the major LHCII complex, whilst

the others were named a, c, d, e or CPs (chlorophyll proteins). All these complexes

formed LHCII antenna. The amounts of newly discovered LHCII complexes were much

lower than those of the major, trimeric LHCII, therefore they have been named ‘minor’

light harvesting complexes or just ‘minor antenna’. They are also referred to as mono-

meric LHCII complexes, since they do not form stable oligomers as LHCIIb. It is worth

to note that the ‘CP’ name is normally attributed to the complex that is not a light har-

vesting antenna. Since LHCII had been discovered years earlier than the minor antenna

complexes and that their amounts were much lower, than the former it was not clear

whether they have the same light harvesting function, or play a different role in the

photosystem II and even belong to the core complex structure. The new separation

methods mainly used nonionic mild detergents: glycosides (see Figure 3.4). The deter-

gents octylglucoside or dodecylglucoside have a relatively short hydrophobic ‘tail’ and

large polar ‘head’, the cyclic structure of glucose residues. These features make such

detergents less penetrating into the hydrophobic protein interior, hence less harmful and

active in removal of pigments. Thornber’s improved gel system was based on the use of

a small concentration of deriphat produced little free pigment. The chlorophyll a/b

ratios were different for all LHCII complexes. LHCIIb (the major LHCII) Chl a/b was

1.33 corresponding to eight Chl a and six Chl b rather than eight and seven, respectively,

as suggested earlier by Kühlbrandt. The Chl a/b ratio of LHCII a, c and d was 2.3, 1.8

and 0.9, respectively. The new isolation method of Bassi (2000) was flat bed preparative

isoelectric focussing. The advantage of it was that the complexes were obtained in rela-

tively large amounts and easily eluted into the buffer solution. The Chl a/b ratio for the

major LHCII was almost the same as in Thornber’s preparation. For the minor com-

plexes CP29 (LHCIIa), CP26 (LHCIIc) and CP24 (LHCIId) Chl a/b the ratios were 2.6,

1.9 and 1.2, relatively close to Thornber’s data. Polypeptides of the minor antenna com-

plexes CP29, 26 and 24 were run at approx 30–31, 28 and 20 kDa, respectively.

Two polypeptides in CP29 complex were often detected. Hence, the use of improved

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separation techniques and mild nonionic detergents enabled the discovery and charac-

terization of all LHCII antenna complexes at the same time. Later preparations that

used modified isoelectric focussing or sucrose gradient isolation techniques revealed

significant variations in the Chl a/b ratio of the minor antenna complexes. For CP29 this

value was reported as high as 3.5 and for CP26 as high as 2.5. The estimated total num-

ber of chlorophylls also varied but consistently reported to be lower than in the major

LHCII. Xanthophyll content and composition were also under debate. The estimates

varied from two xanthophylls to three xanthophylls for minor and from three to four

for the major LHCII monomeric complex. There were several potential causes of such

variations. The first was the low stability of the minor antenna complexes. The second

was the purity of the preparations. Indeed, the major LHCII was much stable and oligo-

meric, whilst the minor complexes were monomeric, more hydrophobic and possibly

belonged to the higher oligomeric or macrocomplex assemblies in vivo , like the photo-

system II complex (see Figure 4.4). In Chapter 9 an intriguing novel role that was

proposed exclusively for the minor LHCII complexes in processes of adaptation of the

photosynthetic light harvesting will be described.

6.3 LHCII Crystallography: The Beginnings

The first structure of the major LHCII complex was published by Kühlbrandt and Wang in

1991. It was a result of electron crystallography study of 2D crystals of the complex. The

resolution was only 6 Å at which they confirmed that the complex is built of three nonco-

valently associated subunits, each containing a polypeptide of approx. 25 kDa with 233

aminoacids (Figure 6.1 a). The shape of the trimer was almost cylindrical with 7.3 nm in

diameter. The elongated shape of the monomer had dimensions of 3 × 5 nm (dashed line in

Figure 6.1 a). For the first time this work visualized three membrane–spanning α helixes,

A, B and C (Figure 6.1 b). The abnormally longer central helixes (4.6–4.9 nm) were slightly

inclined with respect to the membrane normal and appeared to be in contact with each

other. Helix C was found to be oriented more perpendicularly to the membrane plane than

helices A and B (nearly 80 o ). It was shorter, only 3 nm long. Helix B was associated with

the N-terminus facing the stromal side of the membrane and connected through the lumen

loop to the helix C which in turn was connected to the stromal site of the helix A that ended

on the lumenal site of the membrane with C-terminus (Figure 6.1 b). Therefore, the whole

LHCIIb monomer structure spanned the membrane three times as if it was aiming to stitch

it. It was noticed that this structure was very different from the structures of the already

known membrane proteins, bacteriorhodopsin, bacterial reaction centre and matrix porin

and that the only relative complex is likely to be LHCI antenna from PSI, that was recently

discovered and characterized at that time since it has strong sequence homology to LHCIIb.

Each monomer of the structure was found to bind 15 chlorophyll pigments, localized

mainly around central helices A and B in the hydrophobic interior of the complex. The

chlorophylls were identified as densities of around 0.8 nm in diameter and 0.4 nm thick. It

was impossible to distinguish which is chlorophyll a and which is chlorophyll b from this

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structure. In addition the chlorophyll #13 was smaller than the rest and therefore it was pos-

sible that the structure was wrongly assigned. Interestingly, it was found that chlorophylls

are grouped into two layers, one closer to the stromal and the other to the lumenal sites of

the membrane. The upper layer contained eight chlorophylls. Chlorophylls 1 and 5 and 2

and 6 revealed a two-fold symmetry arrangement in the upper parts of helices A and B. This

suggested the existence of conserved chlorophyll binding aminoacids in the homologous

parts of the helixes: a pattern of regularity in pigment arrangement was revealed for the first

time in this study. Chlorophylls of the upper, stromal layer were forming a ring around the

helical structures. All ring structures of chlorophylls were almost perpendicularly oriented

to the membrane plane. It was also predicted that some chlorophylls could have water mol-

ecules involved in their coordination and some aminoacids which did not belong to helical

structures. The authors suggested that the role of the protein in LHCIIb is to ensure highly

Figure 6.1 Structure of LHCII: early days. a: LHCII trimer at 6 Å resolution; b: the side-view of LHCII monomer with numbered chlorophylls and labelled helixes A , B and C . Reprinted by permission from Macmillan Publishers Ltd Kühlbrandt and Wang © 1991.

(a)

(b)

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concentrated chlorophyll binding and correct pigment separation and orientation to enable

efficient energy transfer – the fundamental features of ‘the programmed solvent’ described

in Chapter 5. At this resolution, however, no phytol tails, lipids or xanthophyll pigments

were resolved. It was hard to explain why the complex was in the trimeric form and what

the role of trimerization was. It was commented that the chlorophyll concentration was as

high as 0.7 M and the complex was almost three times more effective in light harvesting that

the soluble light harvesting complex from the bacteria Prosthecochloris aestuarii . The

energy transfer rate (Förster-type mechanism) was predicted by the authors to be very high

in LHCIIb complex, since the interpigment distances were very short. The energy transfer

between chlorophylls localized on different trimers was suggested to be much slower since

the relevant shortest distance between the pigments was found to be longer than for the

chlorophylls localized within one monomer. Still, the structure was lacking many important

details that were essential for creation of accurate energy transfer models for LHCIIb.

Nevertheless, this first structure of the higher plant antenna complex offered novel insights

into the helical and pigment organization of the complex, showing that they are very differ-

ent from the bacterial antenna features, suggesting more efficient and therefore advanced

organization of light harvesting than in prokaryotic organisms.

In 1994, Kühlbrandt and coworkers published a much improved structure of LHCIIb

with 3.4 Å resolution using the electron crystallography on 2D crystals (Figure 6.2 , top).

Almost two times better than previous resolution revealed more interesting features of the

structure. The instrument and crystal quality and the much lower temperature during the

data collection (4 K versus 160 K in the previous work) which allowed better sample

stability were amongst the key factors that improved the special resolution. Lower speci-

men temperature enabled longer data collection times avoiding radiation damage and

allowing better quality information on the high-resolution phases. As a result, it was

concluded that LHCIIb contained not 15 but 12 chlorophyll molecules and two xantho-

phylls. The authors admitted that the structure may be missing two other chlorophylls

that they thought should be present in the structure. The structure revealed only eight

ligands form the central magnesium atom of chlorophylls; aminoacids histidines, gluta-

mates, glutamines, asparagine and glycine. Water was also proposed to play role in the

pigment binding.

Almost 80% of apoprotein residues were successfully fitted to the electron densities

obtained. 36% of aminoacid residues were found to form α helices. More details emerged

regarding the two crossing long transmembrane helices A and B. Pairs of residues F58 and

F173, W71 and L186, M73 and M188 were involved in the two-fold symmetry and charged

R70 and R 185, G65 and G180 formed two pairs bound by ionic interactions in the hydro-

phobic core of the complex (Figure 6.2 , bottom). The ionic forces are the strongest among

noncovalent interactive bonds, even stronger then hydrogen bonds. The presence of such

interactions in the structure of LHCIIb suggests that the hydrophobic core based on the two

helixes is a very tightly-built structure, ensuring stability of the three dimensional structure

of the complex. The new structure revealed the existence of the fourth helix, D, that was

located on the interface between the hydrophobic site of the complex and lumen: clearly an

amphipathic type of helix close to the C terminus.

Rupan_c06.indd 109 8/29/2012 5:37:11 PM


The only criterion of assigning chlorophyll a and b was their location in vicinity of two

found xanthophylls, attributed to luteins. The authors assumed that since the energy in

antenna quickly ends up on chlorophylls a (see Chapter 5 for detail), these chlorophylls

only would need to be protected by carotenoids against photooxidation at the conditions

of high light illumination. Therefore, the seven closest to xanthophyll chlorophylls were

assigned as chlorophylls a and the rest as chlorophylls b . The 3.4 Å resolution was not

a6

a3b3

b2

a4

......GPFSGESPSYLT

DYG WDTAGLSAO P E

T F S K

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A M LA

L

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V

FF

C V F80

70

P E L L

S P N G

G

GEP

40F

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GF

MM

FS

190

RG

LK

N

EK

VK

AE

L

PE

AF

170

60

b1

b5

a1

a7

b5

IS

LAIW

T A130

ML

I

A

VQ

GV

RI

VG

140E

V90K

F GE A V W F K A G S QI F SE

GGLDYLGNPSL V H A

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120

100

110

HL

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210

230220

DPVNN

N AWSYATNFVPGK

G K G T V200

30

50

a5 a2H S R W

D

B A

C

DDALGLPOF S

100G GPY LPD

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GA

150

Stroma

Lumen

Figure 6.2 Structure of LHCII monomer at 3.4 Å resolution. Bottom : schematic presentation of LHCII chlorophyll-binding sites. Reprinted by permission from Macmillan Publishers Ltd Kühlbrandt et al . © 1994.

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enough to distinguish methyl group of chlorophyll a from formyl group of chlorophyll b

(see Figure 5.12). Lutein 1 (L1) was found to be in a close vicinity of three chlorophylls

with the minimum distances of around 4 Å. Lutein 2 was also positioned around three

chlorophylls within similar shortest distances. The symmetry of the two domains of pig-

ments although pronounced is not ideal because of the different protein environment. The

authors proposed that the two luteins, six associated chlorophylls and two transmembrane

helixes, A and B form the central core of the complex. Indeed, lutein was found to be

essential xanthophyll in reconstitution of stable LHCIIb complex from the recombinant

protein. The proposed structural role of xanthophylls in LHCII received here a further

support. Two other xanthophylls, violaxanthin and neoxanthin were proposed to be bound

in to the peripheral parts of the complex. Unfortunately, the resolution did not allow map-

ping of the chlorophyll rings possessing, apart from the four pyrrole cyclic groups, the

fifth cycle (see Chapter 5), neither did it enable seeing the phytol tails. This prevented

determination of the orientation of the transition dipole moments and therefore made

unambiguous calculations of the energy transfer patterns within the LHCIIb complex.

It took another 10 years to reveal the LHCIIb structure at a better resolution. That ena-

bled determination of the orientation of chlorophylls, localization of the phytol tails, find-

ing the lipids as well as missing chlorophylls and xanthophylls. In addition, it was important

to make sure the Chl a and b were correctly assigned. Chang’s group (Liu et al ., 2004 )

published a new LHCIIb structure at 2.72 Å. This was a big improvement that dramatically

influenced and continues to influence the biochemical and biophysical research of LHCII

antenna and beyond. The structure was obtained using X-ray diffraction techniques on 3D

LHCIIb crystals.

6.4 Revealing the Atomic Resolution Structure of LHCII Antenna Complexes

6.4.1 Key Biochemical and Spectroscopic Advances that Aided the Emergence of the Current Atomic LHCIIb Structure

The large gap between the new detailed LHCIIb structure and the old one was filled with

various trials that used alternative approaches to learn more about the LHCIIb structure

than the model of 1994 offered. Work on absorption spectroscopy of recombinant reconsti-

tuted LHCIIb protein aimed to shed some light on identities of chlorophylls a and b . The

idea was to mutate the known pigment-coordinating aminoacids which should allow

removing selectively the chlorophylls. Comparison of absorption spectra of mutated and

original complexes should have enabled, in principle, calculation of the spectra of missing

pigments and if their absorption were to be located around 650 nm the identity would be

Chl b and if 660–680 nm, the pigment should be Chl a . As was described in Chapter 5, the

absorption spectrum of LHCIIb in the red region is indeed represented by chlorophyll b and

chlorophyll a bands. However, the number of these bands is lower than the number of chlo-

rophyll molecules, showing simply that there are pigment forms with similar energies.

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Also, each of the pigments is located in unique aminoacid environment or binding pocket,

such environment is apparently not always causing unique shifts in the absorption spec-

trum of a pigment. This situation is complicated by the fact that pigment-pigment interac-

tions are likely to contribute in some cases to the tuning of pigment energy. In addition to

all these considerations another problem with the reconstitution approach is that removal

of a pigment binding aminoacid may cause alterations in the refolded protein structure that

can affect energies of other pigments. Indeed, the protein is not a rigid building built of

bricks that can be removed without altering its shape. Protein is more delicate and most

importantly mobile structure. Therefore, attempts to identify chlorophylls in the incom-

plete LHCIIb structure were nearly as ambiguous as their tentative assignment based on the

need to photoprotect chlorophyll a in the vicinity of luteins.

More progress using the recombinant technique was made towards the identification of

the xanthophylls’ binding sites. Most notably, the neoxanthin-binding site was predicted by

Bassi’s group (1997, 2000) to be localized in the helix C-domain, on the interface between

helices C and A/B helix domain. It was also established that this xanthophyll was not

essential for the protein folding. Thus only luteins were found to be key core xanthophylls

that were structurally important for the LHCII native folded state. It is therefore not sur-

prizing that for some years it was believed by some that the minor antenna complexes do

not bind neoxanthin and bind only two xanthophylls (see earlier in this chapter). However,

isolated LHCII complexes consistently contained neoxanthin and in case of the major

LHCII, this xanthophyll was in stoichiometric amounts, that is, one molecule per mono-

mer. Furthermore, later it was found that the minor antenna complexes also contain neox-

anthin. Hence, the correct xanthophyll composition seemed to depend upon the LHCIIb

preparation procedure. Recombinant techniques allowed obtaining a membrane complex

in detergent environment, not in the membrane. Hence, the lack of adequate lipids or their

prompt degradation and inability to control appropriate levels of ions are likely to be the

problems of the early reconstitution experiments. In addition, the lengthy purification pro-

cedure could have also been a significant destabilizing factor.

Whilst after all biochemical trials and preparations it became increasingly evident that

there have to be eight Chl a and six Chl b , the xanthophyll composition and conformational

state remained a subject for debate. Core xanthophylls identity and conformation – the two

lutein molecules bound around helices A and B – left no doubt, though. These carotenoids

were in all- trans conformation and essential for the assembly of intact complex with effi-

ciently connected chlorophylls delivering energy towards the terminal emitter cluster of

chlorophyll a . The localization of neoxanthin and violaxanthin was, however, uncertain.

Moreover, it was not clear whether violaxanthin is at all bound to LHCIIb, since the early

preparative work resulted in inconclusive observations, some showing the evidence of sub-

stoichiometrical binding of this pigment and some revealing complete absence of violax-

anthin in LHCIIb. The reconstitution work also suggested that this xanthophyll is not

required for the maintenance of intact tertiary structure of the complex. Hence it was pos-

tulated that violaxanthin is free in the membrane and does not interact with any proteins

apart from the de-epoxidase, that removes from it two epoxy-oxygens converting the mol-

ecule into zeaxanthin: an activity of so-called xanthophyll cycle that will be described in

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detail later. The supporters of free violaxanthin localization in the membrane motivated

their theory that the xanthophylls must be detached from antenna proteins in order to be

readily available for the enzyme. That seemed to be a reasonable explanation. However,

Horton’s group (Ruban et al ., 1999 ) undertook a systematic study that used very mild

detergent solubilization procedure of the PSII membranes and thylakoids and isolated

LHCIIb in somewhat oligomeric state. This preparation was believed to correspond to

more native state of the complex, contained approximately eight LHCIIb trimers and one

violaxanthin per monomer of the complex. Hence, it was proposed that LHCIIb binds eight

Chl a , six Chl b , two luteins, one neoxanthin and one violaxanthin per monomer. The rea-

son for missing violaxanthin in the many early preparations was suggested to be the very

low binding affinity of this xanthophyll. The affinity of violaxanthin binding to LHCIIb

was empirically estimated to be up to five times lower than that of lutein. Therefore, the

trimeric complex should contain four xanthophylls per monomer, with very low-affinity

binding site of violaxanthin. It was proposed that this low affinity should have enabled

accessibility of the xanthophyll for the de-epoxidase. Since the preparations of Horton and

coworkers (Ruban et al ., 1999 ) had virtually no free pigment band (sucrose gradients and

later gel filtrations) it was concluded that the native photosynthetic membrane of vegetat-

ing plants does not contain free pigments in detectable amounts. Later this finding was

confirmed by other research groups.

Towards the end of 1990s there appeared instrumental developments towards analytical

spectroscopic assessment of the state of xanthophylls in the photosysnthetic membrane.

The pioneering work of the laboratory of Robert and coworkers (2004) on the applications

of resonance Raman spectroscopy proved to be a reasonably neat way to address the prob-

lem (for details on the method see Chapters 4 and 8). Violaxanthin was found to change its

configuration from relaxed to slightly out of plane distorted one that resulted in a pro-

nounced alteration of the C—H wagging vibrational mode that became coupled to the

electronic excitation of the molecule. Figure 6.3 (top) displays two distinctly different

spectra of violaxanthin in the 930–980 cm −1 region of the mentioned C—H vibrations

( ν 4 region). In the intact membrane this mode is strong and well-resolved into two bands

(950 and 963 cm −1 ), whilst in the solubilized state (nonionic detergent was added to the

membrane) the spectrum is strongly reduced and the characteristic structure has almost

disappeared. Similar binding ‘fingerprint’ was found in the LHCIIb isolated with the mild

detergent treatment, mentioned in the previous paragraph, when violaxanthin was attached

to the complex in the pigment to monomer ratio close to 1. The resonance Raman spectros-

copy provided independent evidence that confirmed that violaxanthin was an inherent

component of LHCIIb that should have its own specific binding site/domain. In addition,

it was established that this xanthophyll exists in all- trans configuration as two intrinsi-

cally-bound lutein molecules of the complex.

The binding site of neoxanthin and the conformational state of in vivo were not identi-

fied in the 1994 structure. Biochemical work indicated a very strong affinity of binding of

this xanthophyll to LHCIIb suggesting that the binding pocket is not as ‘loose’ as that of

violaxanthin. The conformation of neoxanthin was unambiguously tested by the resonance

Raman spectroscopy. The resonance Raman spectrum of carotenoids in the region of

Rupan_c06.indd 113 8/29/2012 5:37:11 PM


0.0

940 950

Wavenumber, cm–1

Free

960 970

0.2

0.4

0.6

0.8

0.0

Ram

an in

tens

ity, r

el.

Bound Vio

Ram

an in

tens

ity, r

el.

Wavenumber, cm–1

1100 1140

11241132

1157

9-cis

1180 1220 1260

Neo

Figure 6.3 Evidence of violaxanthin binding to the LHCIIb protein in the intact photosynthetic membrane ( top ) and of 9- cis conformation of neoxanthin ( bottom ) obtained by the resonance Raman spectroscopy. The spectrum below neoxanthin (Neo) corresponds to 9- cis violaxanthin of the complex from Cuscuta reflexa, bound into the neoxanthin locus.

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1100–1250 cm −1 (called ν 2 ) is sensitive to the molecular conformation and is broadly used

to establish cis-trans states (see Chapter 3 for structures of xanthophylls). The spectrum

for neoxanthin attached to the light harvesting antenna in the photosynthetic membrane

revealed two specific features that are absent in spectra of all- trans carotenoids. These

features are positioned at 1124 and 1132 cm −1 (see Figure 6.3 , bottom). They are the ‘fin-

gerprints’ of 9- cis conformation. Therefore, not only neoxanthin is consistently present in

LHCIIb of the vast majority of higher plant species it is always in 9- cis conformation (the

complete LHCIIb structure will follow in this chapter). What turned out to be even more

interesting is that in exceptional cases when neoxanthin was found totally absent, it was

replaced by violaxanthin in the same binding pocket and in 9- cis conformation. This fact

suggests that the neoxanthin binding pocket is less strict to the type of the xanthophyll

(violaxanthin can replace neoxanthin, etc.) than the xanthophyll conformation. Hence, it is

a conformation-required binding site. Indeed, Figure 6.3 shows a very much similar spec-

trum of Cuscuta reflexa membranes containing no neoxanthin and enriched in violaxan-

thin. This spectrum is nearly identical to the one above it that belongs to the 9- cis

neoxanthin of spinach. Therefore, biochemical and spectral experiments provided clear

evidence that neoxanthin of LHCIIb complex must be in cis -conformation. The conserved

nature of this state remains to be explained.

6.4.2 The New Structure of LHCII b

Methods that provided fine details of the LHCIIb pigment composition and conformation

were invaluable for those who solved the structure of the complex at atomic resolution.

Chang and coworkers (Liu et al ., 2004 ) first succeeded in providing a very detailed struc-

ture of LHCIIb from spinach showing that it contained all 14 chlorophylls, four xantho-

phylls and two lipids.

Their success was based on development of a gentle isolation procedure that was required

to preserve all pigments in their intact states. They were guided by the previously-men-

tioned biochemical and spectroscopic work that suggested the presence of four xantho-

phylls in LHCII. In addition they succeeded in obtaining 3D crystals of the complex that

could diffract at the highest resolution. The crystallization procedure yielded crystals where

LHCIIb was packed into icosahedral spherical particles each containing 20 trimers. The

outer diameter of the sphere is ~26 nm. It is believed to be embedded into the irregular lipid

bilayer forming a proteoliposome structure. The orientation of trimers is uniform with the

lumenal side always facing into the interior of the sphere. In these particles trimer-trimer

interactions are very minimal and mediated mainly by digalactosyl diacylglycerol (DGDG)

molecules and two pairs of chlorophylls van der Waals forces. Spherical particles contain-

ing LHCIIb interact with each other within the crystal via polar forces established between

hydrophilic outer surfaces. This type of crystals was new and Liu et al . ( 2004 ) named them

Type III membrane protein crystals.

Figure 6.4 represents side and top views on the structure of the LHCIIb monomer. The

layout of the major transmembrane helices, A, B and C is very similar to that of the 1994

Rupan_c06.indd 115 8/29/2012 5:37:12 PM


D-helix E-helix

C-helix

B-helixA-helix

Figure 6.4 2.72 Å resolution structure of LHCII monomer. Top : side view of the monomeric complex. Bottom : top view with the dashed line showing nearly linear alignment of all five α -helices of the complex. Presented in freeware PyMol 0.99.

Rupan_c06.indd 116 8/29/2012 5:37:12 PM


structure, although the latter was done for LHCIIb from pea that had the apoprotein nearly

90% homologous to that of spinach. Lumen side amphipathic helixes D and E are clearly

mapped. The latter, helix E, was not previously observed. Interestingly, the loop between

helices E and C is organized in two antiparallel strands that are stabilized via ionic bonds

from D111 and H120 aminoacids as well as a number of hydrogen bonds: a special detail

worth noticing. Such a structural motif is likely to be of some functional significance yet to

be discovered. The top view shows the linear alignment of all five helices highlighted by

the dashed line.

The complete structure of the monomer is complex enough to see features of individual

domains and pigments. This is largely due to the resolution of nearly all phytol tails of

chlorophylls. Therefore, it makes much sense to present groups of pigments or pigment

domains in order to show and discuss their peculiarities. Figure 6.5 displays all chlorophylls

of the LHCIIb monomer. They are organized in a circle, although quite irregularly. Hence,

one of the major functions of a protein is to make sure that numerous pigments are reason-

ably spaced from each other. The average centre to centre distance is around 10 Å. This

circle of pigments if very tightly arranged around the hydrophobic core of the protein built

by the helixes A and B. Presentation of the LHCII structure using van der Waals surfaces

gives an idea of how compacted all pigments are around the protein (Figure 6.5 , right).

Apart from the resolution of the phytol tails, this structure revealed the fifth cycle of the

chlorine ring making it possible for the first time to unambiguously determine the direc-

tions of the transition dipole moments along X and Y axis of chlorophylls (explained in

Chapter 5). Therefore, this structure offered rich information for the theoretical modeling

of energy transfer and dissipation pathways within the LHCII antenna and therefore was

enthusiastically received by many spectroscopists and theoretical biophysicists. And finally,

the resolution allowed to unambiguously assigning chlorophylls a and b . Indeed, the

C7-formyl group of Chl b could be clearly distinguished from C7-methyl group of Chl a .

Localization of Chl a is shown in Figure 6.6 . Only three of eight Chls a are found closer

to the lumenal site of the complex; Chl a604, 613 and 614. Chls a 610, 611 and 612 form a

Figure 6.5 Chlorophylls of LHCIIb monomer in 2.72 Å resolution structure ( left ) and tight circular way of chlorophyll arrangement around the hydrophobic core of the complex ( right ). Presented in freeware PyMol 0.99. (See Plate 6.5 in colour plate section.)

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terminal emitter locus that receives energy from the rest of chlorophylls within a few pico-

seconds. The excitonic coupling between Chl a 611 and Chl a 612 is about 150 cm −1 , the

highest of all chlorophylls. These pair forms a distinct dimer. Chls a are bound to the pro-

tein via a number of various types of ligands. For example Chl a 611 is only bound into the

complex via the phosphodiester group of phospholipids (PG) that ligates the central mag-

nesium atom. No other specific ligands are found to this pigment. Chl a 610 is ligated by

two aminoacids. Glutamate E180 binds the central magnesium atom and G158 hydrogen

bonds C13 1 carbonyl of the Vth ring of chlorine (see Chapter 5 for the nomenclature). This

Figure 6.6 Chlorophylls a of LHCIIb monomer. Arrangement around the apoprotein ( top ), geometry and nomenclature ( middle ) and types of ligands ( bottom ). Ph: phospholipid. Presented in freeware PyMol 0.99. (See Plate 6.6 in colour plate section.)

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double binding ensures not only the strength but also a robust orientation of the molecule,

essential for the optimal energy transfer efficiency between chlorophylls. In case of Chl

a 602 there are three aminoacids involved in binding: E65, central ligand and Y44 and W46

that both form hydrogen bonds with C13 1 carbonyl. Sometimes water can be involved in the

C13 1 bonding, as in the case of Chl a 603 or can even be a central ligand, as in the case of

the Chl a 604. The structure from Liu et al . ( 2004 ) revealed 70 water molecules present in

LHCIIb monomer, a remarkable number for a small membrane protein.

The reasons why there is a variety of types of ligands for chlorophyll such as aminoac-

ids, lipids and water are not yet understood. Chlorophyll ligands form the core of the bind-

ing pocket that is specifically designed for chlorophylls a and b . Therefore there are no

mixed chlorophyll binding sites, as was previously believed, and the stoichiometry of these

chlorophylls always remains constant. This precision in pigment binding and their accurate

position within binding loci is a manifestation of molecular order ensured by the ‘pro-

grammed solvent’ (see Chapter 5). Coordination interactions and hydrogen bonds are well-

pitched in space, so that the interpigment distance variations are minimal so is the level of

entropy in the whole complex. As was stated in Chapter 1, control of entropy is of a para-

mount importance for life, it is a major attribute of life. The structure of LHCII is a typical

example of molecular determinism, where the genetically programmed apoprotein is capa-

ble of self-assembly, always predictable pigment binding and formation of a tertiary struc-

ture: a remarkable achievement of molecular evolution of life. Yet, the structure is not

frozen. At ambient temperature it is fairly dynamic, as will be shown in Chapter 8. Such

dynamics is a result of remaining entropy that is restricted by the structure of the membrane

protein. This ‘tamed’ entropy is the major driving force behind various nanoscale processes

of life, such as diffusion of molecules in the membrane, their conformational dynamics,

and etc. All owing to a very fine and predetermined redistribution of various weak interac-

tive forces, such as hydrogen bonds and a variety of hydrophobic interactions (see Chapter 1).

Indeed, at 25 o C the energy brought by entropy is about 2.5 kJ mol -1 , this is within the same

order of magnitude as the energy of many hydrophobic interactions and some weak hydro-

gen bonds. Therefore, temperature is such an important factor for life. Indeed, the existence

of the ‘tamed’ entropic energy is absolutely vital for various enzymatic reactions. Hence,

life is not just a frozen order of molecules, it is a rather ‘loose’ order, that enables a certain

level of entropy: a truly existence on the edge that is called non-equilibrium thermodynam-

ics, or thermodynamics of open systems. Organism, organ, tissue, cell, organelle are all

open systems, constantly exchanging energy, substance and information with the external

environment. Therefore, cellular or organellar membranes are full of proteins that fulfill

roles of receptors, gates, transporters: all enabling the vital requirements of life’s micro-

scopic compartments (see Chapter 2).

Chlorophylls b are all localized between helices B and C apart from Chl b 601 that is

bound on the periphery of the complex on the site opposite to helix C near the N-terminal

loop with tyrosine Y24 as a central and only ligand to the magnesium atom (Figure 6.7 ).

Three chlorophylls, b 605, 606 and 607 are located closer to the lumenal site of the com-

plex. Some of these chlorophylls are also hydrogen bound to aminoacids via either C13 1 -

carbonyl or, indeed, C7-formyl: a fingerprint feature of chlorophyll b . However, in Chl

Rupan_c06.indd 119 8/29/2012 5:37:15 PM


b 608 and 609 both groups are involved simultaneously in the hydrogen bonding. Chl b 609

is ligated via magnesium atom to glutamate E139, whilst glutamine Q131 of the C-helix

binds C7-formyl and histidine H68 of the helix B binds C13 1 -carbonyl (Figure 6.7 , bot-

tom). This is a truly remarkable binding arrangement in which two helical structures are

involved. An interesting dimer of chlorophylls b has been revealed by the structure from

Liu et al . ( 2004 ). Chl b 606 and b 607 form a dimer that is bridged by a water molecule that

forms one hydrogen bond with central magnesium atom of Chl b 607 and another hydro-

gen bond with a C7-formyl group of Chl b 606 (Figure 6.7 , bottom). This is an unusual

Figure 6.7 Chlorophylls b of the LHCIIb monomer. Arrangement around the apoprotein ( top ), geometry and nomenclature ( middle ) and types of ligands ( bottom ). Presented in freeware PyMol 0.99. (See Plate 6.7 in colour plate section.)

Rupan_c06.indd 120 8/29/2012 5:37:15 PM


configuration that will be discussed at length along with the role of the other four hydro-

gen bonded formyl groups of Chl b 605, 607, 608 and 609 in Chapter 8.

LHCIIb xanthophylls have fairly fixed binding pockets too. Sometimes they are called

xanthophyll-binding domains that include not only a xanthophyll and aminoacids/helix they

interact with but also closest chlorophylls. Since the reason for the presence of different

types of xanthophylls remains a matter of numerous debates and ongoing investigations it is

common practice to show and discuss relationships between these molecules and their envi-

ronment, most of all chlorophylls, hence the emergence of the domain approach. The

domain of noexanthin is localized in a C-helix region enriched with chlorophyll b

(Figure 6.8 ). It is a fairly polar domain, since Chl b binding pockets have a few polar ami-

noacids or water and neoxanthin itself is the most polar of all LHCII antenna xanthophylls.

Neoxanthin position in the structure is very unusual since the half of the molecule sticks out

of the monomer interior, making it a very peripheral xanthophyll. One would expect a lower

affinity of binding because of this, however, actually it is not the case. Biochemical work

discovered that this xanthophyll is actually very strongly bound into LHCIIb structure. One

of the reasons for this can be seen in Figure 6.8 (right), that shows a hydrogen bond from

tyrosine Y112 to the hydroxyl oxygen of neoxanthin. Another factor could be a 9- cis con-

formation of the end of neoxanthin that is placed in the hydrophobic interior of the complex.

The cis conformation makes neoxanthin more structurally anchored within the hydrophobic

pocket made by chlorophylls and their phytol tails (notably Chl a 604, b608-609). The

whole domain structure suggests that there should be a strong cooperation in pigment bind-

ing overall in the neoxanthin domain. As if neoxanthin is planted into a special ‘cleft’, as

was defined by Chang, formed by a number of chlorophylls and insures their special separa-

tion along with the aminoacids such as L134, M135, V138 (helix C) and W71 (helix B) and

is in a van der Waals contact with this environment (see Figure 6.8 , left). This tight and

complex arrangement of neoxanthin environment explains the very high specificity of this

Figure 6.8 Structure of neoxanthin-binding domain of LHCIIb showing enrichment in Chl b ( left ) and 9- cis end of the molecule hydrogen bonded to tyrosine Y112 ( right ) in the vicinity of the antiparallel strand of the lumenal loop near helices C and E. Presented in freeware PyMol 0.99. (See Plate 6.8 in colour plate section.)

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binding site as was shown in a previous paragraph that discussed the resonance Raman

work. Indeed, this site is rather specific for the 9- cis xanthophyll conformation of a very

polar molecule like neoxanthin or violaxanthin.

Lutein 620 (Lutein 1 in the old nomenclature) is localized in the area of a terminal

emitter chlorophylls a 610-612 (Figure 6.9 ) near helices A and B. It is well-bound to

specific aminoacids localized closer to the stromal and lumenal site by strong hydrogen

bonds via hydroxyl oxygen atoms. On the stromal site, two aminoacids, arginine R162

and leucine L164 from the loop between helices A and C both form these bonds. On the

Figure 6.9 Structure of lutein 620-binding domain of LHCIIb showing the terminal emitter chlorophylls a610-612 ( top ) and hydrogen bonding patterns to hydroxyl oxygens of the xanthophyll ( bottom ). Presented in freeware PyMol 0.99. (See Plate 6.9 in colour plate section.)

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lumenal site, amonoacids proline P205 (helix E) and glutamine Q197 (helix A) form

similar hydrogen bonds to the hydroxyl oxygen of the β -ring. Numerous site chain ami-

noacid residues of helices A and B form a hydrophobic groove that accommodates lutein

620 and holds it by various van der Waals interactions. Hence, as all chlorophylls and

neoxanthin, this lutein is bound by the two types of forces; hydrophobic and hydrogen

bonds. The latter are likely to be a dominating binding factor that holds lutein at an angle

of 31 o with respect to the membrane plane. The hydrophobic groove assists this position-

ing and is likely to ensure efficient self-assembly of the protein during the assembly in vivo or reconstitution procedure in vitro (see Chapter 4). Since the both luteins are found

to be essential for LHCII assembly and stability they must play a crucial structure-stabi-

lizing role for the complex.

Lutein 621 (Lutein 2 in the old nomenclature) is positioned in a similar manner to

Lutein 620 but on the other site behind helices A and B in a similar hydrophobic grove

made by the site chain aminoacid residues and anchored by the hydrogen bonds from

aminoacids localized on the stromal (n-terminal loop aminoacids N47, W48 and A49) and

lumenal (lumenal loop tryptophan W97 between helixes C and B) (Figure 6.10 ). This

binding arrangement ensures ~33 o tilt of this xanthophyll with the respect to the mem-

brane plane. Hence. we observe symmetry in arrangement of the two luteins around heli-

ces A and B giving a very robust structure of the hydrophobic core of the complex.

Interesting feature of Lutein 621 is that it is localized on the inner site of the monomer.

When the complex is in the trimeric state the ε -ring end of this lutein is facing Chl a 603

that is localized on the neighbouring monomer. The chlorophyll is positioned in such a

way that all its five oxygen atoms are directly facing the ε -ring. Such arrangement is

likely to exert a strong field effect upon lutein 621 with consequences on its absorption

spectrum that will be discussed in Chapter 8.

The fourth xanthophyll is localized closer to the monomer-monomer border; it is in all-

trans conformation and unlike the rest of the xanthophylls does not form any hydrogen

bonds with the protein or other pigments (Figure 6.11 ). Unlike other xanthophylls it forms

a relatively large angle to the membrane plain, 56 o : nearly twice bigger than that formed by

other xanthophylls. The binding pocket is mainly hydrophobic, formed by the phospho-

lipid, Chl b 601 and Chl a 613-614 and hydrophobic aminoacids, most notably, on the

C-terminus of the protein, tryptophan W222 and Phenylalanine F228. Since this xantho-

phyll revealed slight differences in the densities of its end groups it was originally assigned

to antheraxanthin: an intermediate of the xanthophylls cycle, essentially a molecule that is

half violaxanthin and half zeaxanthin (see Figure 5.13 from Chapter 5). Later, it was con-

cluded, that since the LHCIIb preparation that was used for crystallization was mainly

enriched in violaxanthin, the xanthophyll structure represents a mixed binding of violaxan-

thin and antheraxanthin. The latter fact implies that de-epoxidation on at least one end

group of violaxanthin does not change the localization of the xanthophylls within the bind-

ing pocket. Although, for the de-epoxidation of the lumenal-site facing ring the removal of

the xanthophyll would likely to be necessary. The xanthophyll cycle activity will be dis-

cussed further in detail in Chapter 9. Interestingly, although violaxanthin is closely sur-

rounded by chlorophylls b 601, a 613 and a 614 it is unlikely to be energetically coupled to

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them due to unfavourable mutual orientations of the transition dipole moments. This notion

has been confirmed by Breton and coworkers (Caffari et al ., 2001) who used the method of

excitation fluorescence spectral measurements (see Chapter 5) to show that violaxanthin

does not play the light harvesting function in isolated LHCIIb complex.

Figure 6.10 Structure of lutein 621-binding domain showing the domain chlorophyllsa 602-604 ( top left ), hydrogen bonding patterns to hydroxyl oxygens of the xanthophyll ( top right ) and interaction with Chl a603 localized on the neighbouring LHCIIb monomer ( bottom ). Presented in freeware PyMol 0.99. (See Plate 6.10 in colour plate section.)

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Figure 6.11 (bottom) displays the arrangement of all four xanthophylls in the LHCIIb

monomer. There is a certain degree of symmetry in it. The top view clearly displays this

idea. The position of luteins relative to helixes is nearly symmetric, supporting the super-

coil helical structure and building in fact a mixed supercoil of the helical and polyene

structure. Neoxanthin and violaxanthin are clearly peripheral, positioned on the opposite

ends of the complex and asymmetric, mainly due to more perpendicular orientation of

violaxanthin relatively to the membrane plane. Unlike the other xanthophylls, violaxanthin

is positioned far away from membrane-spanning helixes and their hydrophobic grooves

that provide van der Waals binding forces that accommodate xanthophylls in the binding

Figure 6.11 Structure of violaxanthin-binding domain of LHCIIb showing interactions with chlorophylls a 613-614 ( top left ), Chl b601 ( top right ). Below are shown the side ( left ) and top ( right ) views of all LHCIIb xanthophylls arrangement within the complex. Presented in freeware PyMol 0.99. (See Plate 6.11 in colour plate section.)

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pocket. Such arrangement of violaxanthin as well as the absence of any hydrogen bonds

with the protein environment can explain why the experimentally-determined affinity of

binding of this xanthophyll is the lowest of all LHCIIb carotenoids and why it took some

time to develop gentle membrane solubilization procedures to preserve it from being lost

from the complex. The story of violaxanthin binding to LHCIIb demonstrates difficulties

in structural research of membrane complexes isolated from their natural environment.

Differential affinity of cofactors’ binding is likely to yield controversial results and delay

the progress towards understanding the complete structure of a membrane protein.

Figure 6.12 shows localization of phospholipid (PG) within the structure of LHCIIb. It

is positioned on the interface between the monomers. The bulky hydrophobic structure

forms large hydrophobic contact surface with the protein and chlorophylls of the locus.

This lipid is essential to for trimerization of the complex. Phospholipase treatment that

removes PG was shown to effectively cause monomerization of trimers. Trimeric structure

of LHCIIb ensures a certain order in positioning of all its 42 chlorophylls. Twenty-four

chlorophylls positioned closer to the stromal surface of the membrane form two circular

rings, highlighted in Figure 6.13 . The inner ring made by Chls a 602 and a 603 ensures effi-

cient intermonomer energy transfer, whilst the outer ring collects energy from all chloro-

phylls of the complex, focuses it on the terminal emitter pigments, Chls a 610-612, which

are ready to give it to the neighbouring complexes: LHCs or, indeed, core photosystem II

antennae (see Chapter 3). Lumenal site facing chlorophylls are likely to collect the absorbed

energy and transfer it upstream to the stromal site facing pigments.

6.5 Structure of a Minor LHCII Complex CP29

Recently Chang’s group (Pan et al ., 2011 ) has produced a structure of another LHCII com-

plex, CP29 or LHCIIa. This is one of the minor PSII antenna complexes that is localized

between LHCII trimer and the inner antenna complex CP47 and is always isolated in a

Figure 6.12 Localization of phosphatidylglycerol (PG) within LHCIIb trimer.PG is shown in black and lighter shading. Presented in freeware PyMol 0.99.

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monomeric state. As was mentioned earlier in this chapter, the minor antenna complexes

CP24, 26 and 29 have been discovered, characterized and isolated later than the major

LHCIIb trimeric complex. Since the amount of them is much lower than the amount of

LHCIIb and they tend to be much less stable than the latter, the biochemical characterization

and structural work has not progressed that far as for the major light harvesting complex.

Chang’s preparation was largely based on the preparative ion-exchange chromatography

approach first applied by Henrysson and coworkers (1989) for the isolation of CP29. These

group actually first isolated this complex in preparative amounts and characterized it

biochemically and spectroscopically. Large quantities of the isolated complex and very high

purity were key factors that enabled Chang to obtain crystals that diffracted at 2.8 Å (Liu

et al ., 2004 ). The chromatography procedure and further gel filtration step applied to improve

the sample purity as well as the crystallization routine were in fact very harsh and lengthy

treatments resulted in the cleavage of very large N-terminal domain of about 70 aminoacids.

However, this domain is believed not to be involved in chlorophyll binding and the produced

Figure 6.13 Organization of all pigments of LHCIIb trimer. Inner and outer pigment rings are highlighted. Arrows show the likely directions of energy transfer from the trimer. Presented in freeware PyMol 0.99.

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structure revealed 13 chlorophyll molecules attached, eight Chl a , four Chl b and a mixed

site of a and b at the 610 location, that was assigned to Chl a 610 in the published structure.

The latter is likely to be an uncertainty of not extremely high resolution as well as the effects

on the structure of the preparation and crystallization procedures. Apart from the missing

N-terminal fragment the structure of CP29 apoprotein is remarkably similar to that of

LHCIIb (Figure 6.14 , top). However, all three transmembrane helixes are shorter than those

of LHCIIb, positions and lengths of amphiphilic helixes D and E are slightly altered and

C-terminus is lacking the motif that is involved in trimerization. These differences in the

protein structure affected some pigment binding patterns, but not all. The central ligation by

water of chlorophylls a 604, b 606, b 607 and b 608 is preserved as well as a special dimer of

chlorophylls b 606 and b607 and chlorophylls a 602, 603, 604, 611, 612, 613 and b608.

Nevertheless, the new structure revealed some alterations in pigments. Chlorophylls b 601

and b 605 are missing; chlorophyll a 614 became chlorophyll b 614; chlorophyll b 609 became

Figure 6.14 Structure of CP29 (LHCIIa) complex at 2.8 Å resolution. Top : side and top views of the whole complex; middle : all xanthophylls view; bottom : the putative terminal emitter chlorophylls a . Presented in freeware PyMol 0.99. (See Plate 6.14 in colour plate section.)

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a 609 and new chlorophyll a 615 is present at the stromal site of the complex near the termi-

nal emitter locus. This chlorophyll strongly interacts with Chl a 611 forming a close to

H-type dimer, where the centres of chlorine rings are positioned at the record close distance

of only around 7 Å (Figure 6.14 , middle). Now, only two chlorophyll a s, a 604 and a 613, are

localized closer to the lumenal site of the complex. The chlorophyll b population is mainly

positioned near the C helix as in LHCIIb. Only Chl b 614 is moved out of this locus.

The CP29 structure revealed only three xanthophyll molecules, neoxanthin, lutein at 620

location (lutein 1) and violaxanthin at 621 location (lutein 2), replacing lutein 621. The latter

fact is consistent with the previous biochemical evidence. Neoxanthin was found in 9- cis

conformation but more bent than the one of LHCIIb due to rotation around C29 atom result-

ing in a smaller angle between its transition dipole moment and the membrane plane (~30 o )

(Figure 6.14 , bottom). Without the fourth xanthophyll the whole structure of the complex

looks less symmetric than that of LHCIIb. Despite the interesting alterations revealed in the

structure of CP29 and novel details described above, the work left a few unanswered ques-

tions. First is related to the missing N-terminal fragment. It is very large in comparison to the

analogous structures of all LHCII polypeptides and is likely to play a specific yet unknown

role. In addition, the fourth xanthophyll is missing in the structure despite the evidence that

is based on the carotenoid to chlorophyll ratio determinations. The latter suggested this ratio

to be around 0.29, corresponding to four xanthophylls and 13 chlorophylls per CP29 com-

plex as opposed to only 0.23 when there are only three xanthophylls. Furthermore, the rela-

tively high enrichment in chlorophyll b and the absorption spectrum of crystallized CP29 are

not entirely typical for previously reported CP29 preparations, that suggested rather lower

relative content of Chl b . Interesting, however, remains the fact the violaxanthin is bound to

lutein 621 binding pocket. The fact that violaxanthin assumes very effective energy transfer

connectivity to chlorophylls a 602 and 603 and b 606-607 makes it very different from the

violavathin bound to V1 site in LHCIIb. In addition, in CP29 violaxanthin is much stronger

bound to the complex as was discovered in the early biochemical and recent theoretical

modelling work. Therefore, often the question arises of a likelihood of poor availability of

violaxanthin to the enzyme de-epoxidase converting it into zeaxanthin: a key mechanistic

event in the major photoprotective mechanism of PSII (see Chapter 9).

6.6 Comparison of LHCII Structure with the Structure of a Simpler Light Harvesting Complex from Purple Bacteria, LH2

There is a variety of different types of light harvesting complexes in different classes of pho-

tosynthetic organisms. Diversity of antenna types is a result of diversity of types of light

habitats of these organisms. Hence, antenna evolution is a result of constant adaptive selec-

tion processes caused by the simple fact that life forms tend to constantly proliferate in space,

conquering various niches and expanding the borders of conditions suitable for life. In this

respect it is interesting to compare the design of the light harvesting complex of one of the

most oldest and well-studied photosynthetic organisms, a purple bacteria Rhodobacter sphaeroides with the structure of LHCII complex. Figure 6.15 shows the monomeric unit of

Rupan_c06.indd 129 8/29/2012 5:37:18 PM


LH2 complex from this organism. This complex forms a peripheral antenna, as LHCII. The

unit is built of two noncovalently associated apoproteins of much lower molecular weight as

the one of LHCII. In weight, α and β polypeptides are only ~5.8 and ~4.8 kDa, respectively,

as opposed to ~28 kDa of LHCII polypeptide. Both polypeptides form transmembrane

α -helixes that are very hydrophobic. The α -polypeptide also forms a small amphipathic helix

Figure 6.15 Structure of a monomeric unit of LH2 antenna complex from Rhodobacter sphaeroides at 2.5 Å resolution. Top : different site views of LH2, a and b indicate the polypeptides of LH2. Bottom : pigment cluster of LH2: carotenoid rhodopin glucoside (RG), 1 Chl800 and 2 BChl850. Presented in freeware PyMol 0.99.

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at its C-terminus. Each unit of LH2 binds only three bacteriochlorophylls, one absorbing at

800 nm (BChl800) and two absorbing at 850 nm (BChl850). There is only one carotenoid,

rhodopin glucoside that is, in fact, a detergent with a long conjugated hydrophobic carbon

chain (see Chapter 3). Therefore the carotenoid is an asymmetric molecule that spans the

membrane and takes part in stabilization of interaction between the two polypeptides of LH2.

All three chlorophylls form a tight special association with rhodopin glucoside’s hydropho-

bic end assisted by their phytol tails (Figure 6.15 , bottom). Such close positioning of these

molecules ensures energy transfer between them and the carotenoid. The arrangement of two

BChl850 resembles of that of a J-type dimer, since their ring structures only partially overlap.

Another striking feature of pigment positioning is that the orientation of the BChl800 ring (as

well as the transition dipole moment) is almost parallel to the membrane plane. Therefore,

even at the level of the monomeric unit one can see signs of a specific pigment order within

the LH2 complex. The order becomes apparent when the structure of the whole oligomeric

LH2 unit is considered. Figure 6.16 shows the structure of the ring that is formed by nine

Figure 6.16 Structure of a complete oligomeric unit of LH2 antenna complex from Rhodobacter sphaeroides at 2.5 Å resolution. Top : LH2 apoprotein; bottom : BChl800 ( left ) and BChl850 ( right ) pigment order. Presented in freeware PyMol 0.99.

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LH2 monomeric units. Its two polypeptides form two concentration circles; the outer one is

formed by β −polypeptide and the inner by the α . The resulting structure looks very sym-

metrical, more symmetrical then the structure of LHCII trimer. The pigment arrangement is

symmetrical too. Nine BChl800 molecules are arranged in a ring with the equal distances

between them of ~21 Å: a distance for efficient Förster-type energy transfer. Not only the

distance but the orientation of the transition dipole moments is all perfectly uniform and

organized for the nearly optimal energy transfer rate. These features of BChl800 are ensured

by the coordination of magnesium with formyl methionine residue, N-terminal aminoacid of

α −polypeptide and additional robustness is achieved by the hydrogen bond from arginine

R20 of the β −polypeptide.

The order in the arrangement of 18 BChl850 is even more striking. The molecules are

well positioned within the interior between the two concentration circles of α − and β −

polypeptides and firmly coordinated by two histidine residues located on each of these

polypeptides. In addition, the ring carbonyls are hydrogen-bonded by tryptophan W45 of

the α −polypeptide, ensuring further robustness in orientation and positioning. As a result a

very symmetric crown-type arrangement of BChl850 is achieved, enabling a very fast, sub-

picosecond energy transfer between these pigments.

The whole structure of LH2 pigment population looks very well-ordered, more than

that of the LHCII, where the symmetry is held only in the supercoiled helices A and B

and is broken by the introduction of helix C and less ordered pigment binding. The sizes

of LH2 oligomer and LHCII trimer are very similar (~8 nm in diameter each). However,

LHCII is not a hollow particle as LH2. The interior is filled with pigments that form a

far from being symmetric inner circle. The total number of LHCII trimer bound pig-

ments is 54, including carotenoids, whilst LH2 binds only 36 pigments (50% less). On

the other hand, the total molecular weight of the polypeptides required to organize LH2

and LHCII pigments is approximately the same (~90 kDa). It seems that the higher

plants managed to pack more pigments into the light harvesting complex. In addition, it

takes only a few to form a trimeric structure as opposed to the nanomeric one of the LH2.

The structure of LHCII is less symmetric and hence less organized than the one of LH2.

One of the likely explanations is that there was a pressure to increase the cross-section

of antenna without the increase in the space it took in the membrane. The increase in

pigment concentration is a risky venture due to the danger of the concentration quench-

ing that leads to very fast energy dissipation within concentrated pigments, hence

undermines the efficiency of light energy storage within the antenna and the photosystem

efficiency as a whole (see Chapter 5). The nature had to balance between the cross-section

size and the efficiency of the placed pigments at high concentration. The symmetric

arrangement that was utilized by early evolution in purple bacteria has reached its limits

and therefore other solutions have been tried. As a result, the evolved LHCII came up as

less ordered structure, but more solid for it contained a fewer apoprotein subunits,

binding more pigments per volume and with ability to flexibly control its efficiency

(see Chapters 8 and 9). Hence, the increased entropy in the light harvesting design

was a positive evolutionary achievement, that offered more adaptable system, providing

the reaction centre with the right amount of energy required for photosynthesis and the

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organism as a whole. As will be shown in Chapter 9, the rather careful light harvesting

is indeed vitally required for oxygenic photosynthesis.

References

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‘This … whatever-it-was … has now been joined by another … whatever-it-is … and they are now proceeding in company .’

Winnie-the-Pooh (by Alan Alexander Milne)

The previous chapter described in atomic detail the structures of the major and one of the

minor light harvesting complexes of photosystem II. This chapter is dedicated to

the equally-important theme of structural integration of the photosynthetic antenna within

the photosystem. Photosystem II, the heaviest and evolutionary the youngest complex

in the photosynthetic membrane, contains the most convoluted light harvesting antenna.

This chapter shows how the combination of structural and biochemical research has

evolved our current knowledge of the macrostructural organization of the photosynthetic

unit. It has been some time since the photosynthetic unit concept was put forward; how-

ever, it took a few decades to unravel its structure with the precision necessary to under-

stand how the functions and adaptive mechanisms are built in. As was discussed in detail

in Chapter 3 the multi-subunit organization of the photosynthetic membrane complexes is

vitally important for sustaining their optimal ‘working regime’ and is an inevitable conse-

quence of a sequential character of the energy transformation events in the light phase of

photosynthesis. Lowering the entropy of the system; the ability to bind, stabilize and coor-

dinate the cofactors; the requirement for concerted and precise performance of the energy

transformation reactions; the multifunctional performance and the fulfilment of the regula-

tion requirement; are all essential advantages and features of a macrostructural multi-

subunit organization. The processes of photon absorption, excitation energy delivery to the

reaction centre, primary and secondary charge separation and rereduction of the oxidized

Structural Integration of Antennae within Photosystems

7

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donors are all taking place and fairly coherent within the macrostructure of the photosystem II.

The heterogeneity of light harvesting antenna complexes and the sheer variety of antenna

types in comparison to the reaction centres are indicative of the complexity, specialization

and vital importance placed by evolution on light harvesting processes in photosynthesis

as a primary source of energy for life.

7.1 Light Harvesting Complexes Gene Family

In parallel to the development of biochemical analytical and preparative procedures of

separation and isolation of various LHCII complexes the work on identification of genes

that encode their apoproteins was underway in 1980s. By the middle of 1990s it became

clear that the entire family of nuclear genes exists that encodes a variety of polypeptides of

light harvesting antenna complexes and related proteins of the thylakoid membrane.

Progress in the Arabidopsis genome sequencing revealed at least 15 genes encoding vari-

ous LHCII antenna complexes. This offered rich information about the aminoacid sequences

of LHCII polypeptides that greatly helped the work on LHCII structure determination as

well as understanding of the folding process into the native structure with bound pigments.

Three genes encoded the polypeptides of the major LHCII, Lhcb1, 2 and 3, were identified

called Lhcb1, 2 and 3 (gene name is italicized). It was revealed that homotrimers of LHCII

exist, encoded by Lhcb1 as well as heterotrimers, encoded by Lhcb1 and 2 or Lhcb1 and 3

genes. In fact there are five types of Lhcb1 ( Lhcb1.1, 1.2, 1.3, 1.4 and 1.5 ) and four types

of Lhcb2 genes ( Lhcb2.1 , 2.2 , 2.3 and 2.4 ) that have small sequence variations. The level

of their expression varies a lot, which is not yet explained. In contrast to Lhcb1 and 2 gene

groups there is only one type of Lhcb3 . The assembly of LHCII complexes is a process that

requires N-terminus truncation of the precursor protein sequence. Hence, after such pro-

cessing, mature proteins that are encoded by Lhcb1.1, 1.2 and 1.3 are identical. Lhcb1.4

and 1.5 proteins are different, but only by a small number of aminoacid residues. They are

called Lhcb1 polypeptide isoforms. The role of such variations is not yet clear. Existence

of multiple gene copies makes it difficult to perform gene manipulations, like antisense or

knock-out as described in Chapter 4.

The minor light harvesting antenna genes, Lhcb4 (CP29), Lhcb5 (CP26) and Lhcb6

(CP24) encode Lhcb4, 5 and 6 polypeptides; the apoproteins of CP29, 26 and 24 com-

plexes, respectively. There are three gene copies of Lhcb4 and only one for each, Lhcb5

and 6 . The expression levels of Lhcb4.1 and 4.2 are more than an order of magnitude higher

than those of Lhcb4.3 . The polypeptide sequences of LHCII complexes reveal different

levels of homology. Lhcb1-2, the polypeptides of the major LHCII complex, have closer

similarity than the isoforms of Lhcb4 polypeptides (CP29). The latter is more homologous

to the Lhca subfamily that encodes polypeptides of LHCI light harvesting antenna of PSI.

CP24 polypeptide (Lhcb6) also bears homology to Lhca group. The closest relative of

Lhcb1-3 genes is Lhcb5 that encodes CP26 polypeptide. Interestingly, this polypeptide

possess WYGPDR N-terminal motif that was found essential for the formation of the trim-

eric light harvesting complex structure. Hence, CP26 can potentially form trimers as indeed

Rupan_c07.indd 136 8/29/2012 5:36:46 PM

Structural Integration of Antennae within Photosystems 137

would be shown later in this chapter. Recent studies of the Lhc gene superfamily have sug-

gested the existence, apart from mentioned 15 Lhcb genes, of the four genes of Lhca -

encoding polypeptides, Lhca1-4 that form the four types of PSI light harvesting antenna

complexes. The LHCI antenna polypeptides are related to Lhcb subfamily. Their closest

relatives from the latter are Lhcb4 and 6 of CP29 and CP24 complexes, respectively. Lhca

polypeptides all possess three transmembrane helixes. The additional light harvesting fam-

ily genes are so-called rarely-expressed genes and may play a role different from the abun-

dantly expressed ones. Genes like Lhcb7 (the most analogous to Lhcb5), Lhcb8 (formerly

Lhcb4.3 isoform), Lhca5 and Lhca6 have been recently mentioned in the literature.

Abundance of Lhc polypeptides as well as the existence of multiple subunits of inner

antenna and reaction centre components pose an obvious question: how are they all bound

to each other and what is the shape and significance of the structures that constitute the

photosynthetic unit?

7.2 Toward the Structure of a Complete Photosystem II Unit: Supercomplexes

Work of Staehelin , Miller and Simpson in early 1970s (see Staehelin, 2003 ) on the investi-

gation of the photosynthetic proteins using the freeze-fracture electron microscopy revealed

existence of densely-redistributed particles of various sizes that belonged to photosystems

I and II (similar to those shown on Figure 3.1). The particle areas varied from about 50 to

200 nm 2 . With the largest size identified as corresponding to the PSII complex. It became

clear that in vivo photosystems existed in the form of multi-subunit complexes. However,

freeze-fracture technique could not provide even basic crude structural details of these

macrocomplexes mainly due to the limited resolution (~5 nm) mainly arising from the

thickness of platinum replicas that were actually looked at (see Chapter 4). Even in case of

rarely found crystalline arrangements of PSII particles in the photosynthetic membrane,

only a few structural elements of the particle can be seen using this method. Figure 7.1

shows a fragment of a replica displaying ordered arrays of PSII particles. Arrows point

towards the four structural features within the particle that are relatively regularly repro-

duced. Nevertheless, other particles have less clear symmetry and a fewer if any features.

Hence, whilst the freeze-fracture technique is excellent for the studies of redistribution of

membrane complexes in intact photosynthetic membrane (see Chapter 9) it is not of suffi-

cient resolution to enable detailed structural characterization on the level of even the fairly

large PSII complex.

An obvious conclusion from the lack of adequate methods to map the structure of

membrane-bond photosystem complexes was to separate them from the membrane as indi-

vidual particles. The quest for isolation and characterization of antenna complexes, described

in Chapter 6 revealed the evolution of related biochemical approaches. The fundamental

feature of all of them was the type and concentration of detergent used to solubilize the

membrane and keep hydrophobic membrane proteins in solution. Several types of detergents

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were used. Initially, ionic detergents SDS and deriphat as well as the nonionic detergent

Triton X100 were employed for this purpose. Unfortunately, these detergents were too harsh

to preserve some pigment binding and oligomerization states of photosynthetic complexes.

Ultracentrifugation or ‘green’ gel electrophoresis techniques that were used for separation of

different complexes normally produced significant amounts of free pigment bands and mono-

meric proteins. For the purposes of purification of individual photosystem components these

approaches were almost fine, apart from the fact that they removed pigments from the com-

plexes hence affecting their true pigment composition. Ionic detergents, in particular, could

remove pigments even at relatively small concentrations under conditions where solubiliza-

tion of proteins was still incomplete. Hence, even for the analytical purposes the search for

a milder solubilization routine was an essential step. Digitonin was another detergent that

was used in the early days of the photosynthetic membrane solubilization. However, its

primary application was to separate different membrane fragments containing photosystems

I and II (see Chapter 6). The molecule of this detergent is very large and contains a group of

glycosidic residues as well as hydrophobic steroid structure. Its solubilization action is con-

sidered to be mild and therefore appropriate for incomplete membrane solubilization at

certain conditions and therefore preservation of membrane fragments. However, even in the

early 1970s some work emerged that used digitonin in combination with the polyacrylamide

gel electrophoresis. Fradkin and coworkers ( 1972 ) reported the existence of heavy pig-

mented protein bands, larger than CP1 and 1a photosystem I bands on Anderson SDS elec-

trophoresis. Hence, it was demonstrated that it was practically possible to sustain the

intactness of highly oligomeric complexes, predominantly PSII, in an isolation procedure.

The next important step in the isolation of oligomeric intact PSII was synthesis and

application of a range of mild glycosidic detergents like octylglucoside (n-octyl- β -D-

glucoside), containing one glycosidic residue (see Figure 3.4) and dodecylmaltoside

Figure 7.1 Freeze-fracture electron microscopy image of the fragment of ordered photosystem II complexes in the photosynthetic membrane. Arrows indicate structural features within a single complex. Bar corresponds to a 50 nm scale.Courtesy of Tomasz Goral.

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(dodecyl- β -D-maltoside and dodecyl- α -D-maltoside), containing two glycosidic residues

as a hydrophilic part and a hydrophobic saturated chain of eight or 12 carbon atoms. These

detergents were smaller and simpler molecules in comparison to digitonin and possessed

relatively short hydrophobic tails. Bassi (1987) used octylglucoside to solubilize PSII-

containing membrane fragments (known as BBY particles, see Chapter 4) and obtained

oligomeric PSII complexes containing reaction centre, core antenna and light harvesting

antenna complexes. It was found that some interactions, particularly those between core

antenna proteins, CP43 and CP47 as well as LHCII and the core antenna were stabilized

via ionic interactions with participation of magnesium cations. Other complexes, such as

CP24, 26 and 29 were associated with PSII via hydrophobic interactions. Therefore, it was

established that not only the weakest, hydrophobic forces (van der Waals) are needed to

maintain the macrostructure of PSII but some stronger, ionic interactions as well. This

work prompted researchers to experiment with glycosidic detergents in order to establish a

procedure that could eventually produce relatively stable photosystem II complex with the

size of antenna matching that of the functionally-determined photosynthetic unit, that is,

~250 chlorophylls per one RCII. Generation of stable PSII macrocomplexes was essential

for structural analysis using either electron microscopy with the single particle image

enhancement analysis or crystallographic studies (see Chapter 4). Detergent solubilization

of the membrane fragments containing PSII was normally followed by either sucrose gra-

dient ultracentrifugation (see Chapter 4, Figure 4.1) or gel filtration procedure. The latter

became more common since it was relatively fast, reproducible and did not require large

amounts of sample. Figure 7.2 shows a typical gel-filtration chromatogram of separation of

160.0

0.3

0.6

Abs

orpt

ion

at 6

70 n

m, r

el.

0.9

1.2

1.5

1.8

18 20 22 24 26

Elution time, min

28 30 32 34 36

PSII

PSIPSIIcore

LHCIItrimer

LHCIImonomer

Figure 7.2 Gel filtration elution profiles of solubilized photosystem II membranes (BBY particles) and isolated LHCII trimers and monomers. Arrows relate the elution time of separately run purified LHCII trimers and monomers.

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various pigment-protein complexes associated with the PSII-enriched membranes (BBY

particles). The method uses porous gel that traps and slows down the movement of smaller

particles and leaves free large ones. Therefore it works like filtration in reverse, trapping

the smaller and letting the larger complexes elute faster. Therefore, the fastest moving are

oligomeric PSII complexes or supercomplexes (PSII, 19 min elution time). Some minor

presence of PSI complexes is also detected by the shoulder at ~22 min elution time. Smaller

PSII core complexes devoid of light harvesting antenna are eluted at 24 min. Next are

eluted even smaller LHCII trimers followed by monomers (27 and 30 min elution times,

respectively). Denaturing polyacrylamide gel electrophoresis is normally used to analyse

the polypeptide compositions of all fractions. The oligomeric PSII band is identified as

containing minor and some major LHCII complexes apart from the core PSII complexes.

However, since there is significant amount of separate LHCII trimers it is possible that not

all of them are associated with the PSII fraction.

The first single particle images of oligomeric PSII fractions from higher plants using

dodecyl- β -D-maltoside for solubilization of membranes enriched in PSII (BBY frag-

ments/particles) were obtained by Boekema in mid-1990s. Initially, sucrose gradient sepa-

ration procedure was used, which later was replaced by the gel filtration method (Boekema,

1995 ). Figure 7.3 shows the first image of an oligomeric PSII particle (supercomplex) that

contained RCII, core antenna, minor and trimeric LHCII complexes and was ~700 kDa.

The dimensions were ~27 nm long and ~12 nm wide, that corresponds to the area of ~320

nm 2 . This is more than 60% larger than the average reported area of the PSII particles on

EFs freeze fracture membrane surfaces (see previous), meaning that the freeze-fracture

method did not reveal peripheral light harvesting complexes and showed mostly RCII core

complexes. Indeed, LHCII was found to be localized on the complimentary, PFs surface

of the grana membranes (see Chapter 4, Figure 4.5). Remarkably the supercomplex struc-

ture revealed dimeric organization of the photosystem. Further investigations showed that

this particle, named the PSII supercomplex, lacked one of the minor LHCII antenna pro-

teins, CP24. The complex was named C2S2 particle, meaning it contained dimeric core

(C) and two LHCII trimers (S), strongly attached to the core via the two copies of two

types of the minor antenna complexes, CP26 and CP29. The localization of the core com-

plex proteins in the supercomplex map has been a subject of debates and guesses based

mainly upon work using cross-linkers: binary reagents useful for identification of neigh-

bours in the complex oligomeric structure as PSII. The Figure 7.3 caption describes some

of the structure assignments done on the basis of cross-linking experiments. It became

later apparent that the particles seen on the freeze-fracture replicas of PSII membranes

(EFs surfaces) (Figure 7.1 ) corresponded to C2 or PSII core with probably some minor

LHCII antenna complexes only (see Chapter 9 for more detail). The minor antenna com-

plexes seem to be acting as linkers of the trimeric LHCII to the core antenna, CP43 and

CP47 complexes. Trimeric LHCII was found to be more robust and stable and less hydro-

phobic in comparison to the monomeric (minor) LHCs, suggesting that the latter are more

intrinsic complexes and less independent from the supercomplex structure than trimers.

Whilst the assignment of LHCII trimer locations was not difficult to guess, the assign-

ment of the densities corresponding to the minor LHCII complexes was not strait forward

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due to their intrinsic location and small size. Structural studies on PSII supercomplexes

using the method described previously have only revealed the density areas which matched

the estimated size of a monomeric LHC complex. Biochemical analysis of purified super-

complex preparations showed the presence of CP26 and CP29, and it was therefore assumed

that two areas, on either side of the supercomplex, 5 + 6 and 7 + 8, are occupied by these

two proteins (Figure 7.3 ). Since CP26 and CP29 complexes are predicted to have similar

overall shape, it obviously could not be deduced at which of the two potential sites CP26

and CP29 were located. The assignment of CP26 and CP29 in the complex was therefore

based only on the study of crosslinking products, as was already mentioned above. Clearly,

a new methodology was required to solve the minor antenna assignment ambiguity. This

methodology was the reversed genetics approach of gene antisense technique (see Chapter 4

for details) that enabled to eliminate synthesis of Lhcb5 polypeptide, an apoprotein of CP26.

Jansson successfully pioneered this approach on Arabidopsis in 2001 (Andersson et al ., 2001 ). Figure 7.4 shows the key result of this work. The PSII supercomplex structure was

significantly altered in the antisense plants. The density at the tip of the supercomplex

Figure 7.3 First images of dimeric organization of PSII complex associated with minor and trimeric LHCII. 1–9 are various density areas revealed by the single particle analysis. Area 9 was tentatively assigned to LHCII trimer; areas 5–8: to the minor LHCs; area 4 and 2 to CP43 and CP47 complexes, respectively. Areas 1 and 3 were attributed to D1/D2 reaction centre complex. Reprinted with permission from Boekema et al . ( 1995 ). ©1995 National Academy of Sciences, U.S.A.

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neighbouring to the trimer location was largely absent in the manipulated plants. This den-

sity as originally labelled as densities five and six was proposed to correspond to the miss-

ing CP26 complex. Therefore the densities seven and eight have been assumed to belong to

CP29: all in agreement with the biochemical and cross-linking evidence. Later, creation of

the plants lacking CP24 and CP29 complexes confirmed the previously made structural

assignments.

The most important advance in the early work of Boekema was that the PSII was discov-

ered to be in a dimeric state. However, the knowledge of the complete photosystem struc-

ture with all of the minor and at least four trimeric light harvesting complexes was lacking.

Dekker together with Boekema ( 2005 ) first applied another dodecylmaltoside, dodecyl- α -

D-maltoside, and a more rapid isolation procedure that resulted in partial solubilization of

membranes and generation of a range of oligomeric PSII complexes. The gel filtration

elution profiles they obtained were very similar to that depicted in Figure 7.2 characteristic

of a very pronounced PSII supercomplex band eluted at around 19 min. This fraction con-

tained some particles that were larger than C2S2 complexes and have been assigned to

C2S2M2 particles that contained two cores, two strongly bound (S) and two medium bound

(M) LHCII trimers (Figure 7.5 ). Moreover, the hypothetical reconstituted image of

C2S2M2L2 particle was proposed (L after ‘loosely’ bound LHCII trimer) for the photosys-

tem containing three LHCII trimers per one monomeric RCII complex. Figure 7.5 shows

the image of the largest PSII supercomplex that contained three LHCII trimers per one

reaction centre. Interestingly, the preparations used for this study revealed the presence of

Figure 7.4 PSII supercomplexes (C2S2) from Arabidopsis plants lacking CP26 minor antenna complex: assignment of CP26 localization within the PSII structure (shown by number ‘26’). Scale bar is 10 nm. S is strongly-bound LHCII trimer. Reprinted with permission from Yakushevska et al . © 2003 The American Chemical Society.

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the missing in C2S2 particle minor antenna complex, CP24 that was assigned in the

C2S2M2L2 structure (Figure 7.5 ). The assignment suggests that the binding of this com-

plex depends on the binding of the M trimer, meaning the complex is already present in

C2S2M2 structure.

Even the proposed C2S2M2L2 structure could not explain the full light harvesting

antenna size of PSII that could reach five or sometimes more trimers per one reaction

centre. Further thorough electron microscopic studies of Boekema and Dekker revealed

the existence of yet another macrostructure in the photosystem II containing membranes.

This structure was highly symmetric and suitable for the single particle analysis tech-

nique. Figure 7.6 shows the structure of oligomeric LHCII complex, containing seven

trimers and was called an icosienamer . This structure is a result of averaging of more than

100 projections. It apparently exists in the membrane independently from the supercom-

plex but should be localized in its vicinity in order to enable efficient harvested energy

transfer towards the reaction centre. If, for example, the functional antenna size of PSII

corresponds to five LHCII trimers per one reaction centre or 10 per one dimeric PSII core,

then each C2S2M2 particle should interact closely with approximately one icosienamer.

The icosienamer particle is relatively unstable and may well be of a transient nature due

to the loosely-bound LHCII trimer’s mobility and interactions in the thylakoid membrane.

In general, the existence of loosely bound trimers of LHCII suggests that PSII oligomer

structure is relatively flexible. Such flexibility has important adaptive functional implica-

tions that will be described in more detail in Chapter 9.

After some years of improving electron microscopy instrumental tools as well as perfecting

the methods of preparative isolation and single particle analysis by utilizing the automated

approaches to collect literally tens of thousands of particles, classify them and average the

images, Boekema’s group succeeded in achieving a PSII supercomplex resolution of about

(a) (b)

Figure 7.5 Towards the complete macrostructure of Photosystem II. Discovery of C2S2M2 PSII particle (a) and proposed structure of C2S2M2L2 complex (b). Reprinted with permission from Boekema et al . © 1999 Wiley-Blackwell.

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1 nm that enables to identify separate helices within LHCII trimer and minor antenna com-

plexes (Figure 7.7 ). The C2S2M2 structure turned out to be the most typical and complete

state in variety of preparations and was taken as a base structure of PSII with attached

peripheral antenna components. It carries all minor antenna complexes, CP24, CP26 and

CP29 as well as two LHCII trimers per monomeric PSII reaction centre complex. The helix

contour shape and orientation makes it possible to map the atomic structures of LHCII

monomers and trimers onto the single particle images so that their accurate orientations

within the macrostructure can be determined. This approach is extremely useful for the

theoretical and experimental studies of the patterns and pathways of the energy transfer

between different topological antenna components of the supercomplex. The structural

advances in the knowledge on PSII paved the path for the progress in accurate quantitative

description of essential antenna events: photon absorption, energy transfer and trapping.

Clearly the role of protein in the supercomplex assembly is central: it does not only carry the

essential functional cofactors, like pigments, it enables and sustains correct protein interac-

tions in order to provide optimal pigment orientations and distances for efficient energy

transfer and its possible regulation.

The existence of PSII in the form of a dimer and arrangement of a large light harvesting

antenna around the core complex still remain subjects of intense research. This aims to

understand the significance and advantages of the dimeric state as well as the way in which

the complete major antenna (five or sometimes more LHCII trimers transferring energy to

each RCII) interacts with the core complex, as well as the reason for ‘loose’ arrangement

of the PSII structure in general.

Figure 7.6 Discovery of the heptameric oligomer of LHCII, icosienamer. The LHCII trimer atomic structures are mapped upon the related icosienamer densities. The particle resolution is high enough to enable correct alignment of each trimer within the oligomeric particle. Reprinted from Dekker and Boekema.© 2005, with permission from Elsevier.

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7.3 Supramolecular Structure of Photosystem I : LHCI

Indeed, the structural flexibility of the PSII peripheral antenna binding can be an essential

entropic giveaway in order to making the system more adaptable to the environmental fac-

tors at certain conditions (see Chapter 9). This ‘loose’ arrangement made the work on the

high resolution atomic structure of PSII (or even C2S2M2 structure) very difficult. Only

the structure of cyanobacterial PSII core with the oxygen-evolving complex was recently

solved by Shen and coworkers (Umena et al ., 2011 ) with brilliant detail (1.9 Å resolution)

providing the key fundamental insights upon the electron/proton transformation and trans-

fer pathways in this photosystem as well as the catalytic heart of the water-splitting com-

plex. Attempts to preserve the supercomplex structure during the crystallization procedure

have been so far unsuccessful. Unlike PSII, the PSI supercomplex has revealed a much

better stability and could be almost routinely prepared by the sucrose gradient centrifuga-

tion or gel filtration techniques. Solubilization of unstacked thylakoid membranes with

dodecyl- β -D-maltoside normally results in formation of a dense band on the sucrose

gradient corresponding to ~0.7 M of sucrose (see Chapter 4, Figure 4.1). This band con-

tains the complete PSI supercomplex with its own peripheral light harvesting antenna,

LHCI. Figure 7.8 (a) shows the 4.4 Å resolution structure of higher plant PSI obtained by

Figure 7.7 The latest macrostructure of Photosystem II C2S2M2 complex at near 1 nm resolution (a) with overlaid crystal structures of its components, core reaction centre complex and monomeric and trimeric LHCII complexes (b). Panel (c) displays identification of LHCII helices on the single particle image analysis using the crystal structure of LHCII. S and M are strongly and medium bound LHCII trimers; C is the area of monomeric PSII core complex. Reprinted from Kouřil et al . © 2012 with permission from Elsevier.

(a) (b) (c)

Helix AHelix B

Helix C

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Nelson’s group (Ben-Shem et al ., 2003 ) using the X-ray crystallography. This study

revealed that it exists as a monomeric asymmetric particle of approx. Size of 12 × 15 nm

that contains peripheral LHC antenna complexes. Unfortunately, due to the limited resolu-

tion the details of LHCI complexes are not as well-resolved as those of LHCII and CP29.

Interestingly, LHCI antenna complexes are much tighter associated with PSI core complex

and could only be removed from it using the treatment with multi-ionic detergent

Zwittergent-16. The Figure 7.8 (b) shows separation of LHCI from the core PSI complexes

following the Zwittergent incubation of isolated PSI fraction. The sucrose gradient bands

of PSI corresponding to the intact supercomplex, core PSI devoid from LHCI antenna

complexes, LHCI and free pigment zone can be identified using the denaturing gel electro-

phoresis. The molecular mass of all Lhca polypeptides is slightly lower than that of the

majority of Lhcb proteins and varies from 20 to 25 kDa.

Proteins Lhca1 and 4 form a relatively stable dimer: they are closer positioned towards

each other than Lhca 2 and 3 on the structure. All four LHCI complexes have similar design

to LHCII and bind less chlorophyll b (average Chl a/b ratio is ~3.7), similarly to the minor

LHCII antenna complexes. In addition LHCI binds xanthophylls lutein and violaxanthin, but

not neoxanthin, the most polar carotenoid. Unlike LHCII complexes, LHCI bind β -carotene,

the most hydrophobic carotenoid of higher plant peripheral antenna. As will be shown in the

next chapter, polarity of antenna carotenoids/xanthophylls can affect their structural and

functional dynamic properties essential for regulation of light harvesting efficiency.

Figure 7.8 Structure of the photosystem I supercomplex (a) and separation of LHCI antenna from the core PSI complex (PSIc). Presented in freeware PyMol 0.99 (b). Horizontal top panel shows the sucrose gradient tube with separated PSI components. Fp, free pigment zone. Bottom panel shows the SDS PAAG analysis of each of the sucrose gradient fractions. Numbers on the left indicate the positions of molecular weight markers.

Lhca1

Lhca4

Lhca2Lhca3

(a)

16

30

36

50

64

Fp PSIcPSILHCI(b)

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Another feature of LHCI antenna is that it carries a relatively small number of chloro-

phylls compared to the core complex. Whilst the latter carries nearly 110 chlorophyll mol-

ecules, all LHCI binds only 56, meaning that the PSI light harvesting antenna carries only

30% of a total cross-section of the reaction centre. In case of PSII this number reaches

85%. Clearly, the structures of antenna systems of the two photosystems are principally

different. PSII relies upon the highly mobile and relatively independent peripheral antenna.

PSI reaction centre complex incorporates fused and very large antenna and altogether with

relatively small LHCI is robustly built with much lower levels of entropy and excluding

any potential flexibility of the superstructure. Hence PSI design seems to be more solid and

therefore geared better to stably harvest light than the ‘loose’ structure of PSII. Chapter 9

will present a number of considerations that aim to explain the reasons behind the ‘loose’

design of the light harvesting antenna of PSII in detail.

7.4 Photosynthetic Membrane Protein Landscapes

Staehelin’s work was one of the first that provided visual information on the special segrega-

tion of PSI and PSII complexes using the freeze-fracture electron microscopy (see Staehelin,

2003 and Chapter 4 for details). Alternative approach to visualize redistribution of photosys-

tem complexes in the membrane is the negative stain transmission electron microscopy. The

method requires deposition of incubated in solutions of heavy metal salts membranes upon

the carbon-coated grids and their attachment to the surface so that they can be exposed to the

electron beam perpendicular to the membrane plane. In case of the stacked thylakoids of

grana that are virtually three dimensional this is not a straightforward procedure. Analysis of

FPLC preparations of solubilized grana membranes enriched in PSII often results in appear-

ance of membrane fragments containing often only two appressed membranes. This arrange-

ment is more two-dimensional than the whole grana stack and therefore the membrane

fragments can be relatively easily aligned on the grid for negative transmission electron stain

microscopy. Figure 7.9 (a) shows one of such fragments stained with uranyl acetate showing

relatively ordered arrays of PSII. Due to such order these arrays or crystallinity areas can be

analysed using the approach of single particle analysis applied by Boekema to reveal the

structure of the PSII complex (see previously). Accumulation of a large number of such mem-

brane fragments, their proper alignment and image averaging results in generation of signifi-

cantly resolved PSII structure in the crystalline arrays that resembles the structure of C2S2M2

supercomplex described above. Figure 7.9 (b) depicts an averaged image of a membrane frag-

ment clearly showing the intense PSII core densities with structural features within as well as

the structures of S and M trimers (indicated by arrows). The C2S2M2 particle can be easily

traced (dotted line) and has very similar shape and size to the C2S2M2 image of an isolated

PSII (see Figure 7.7 ). Therefore, the membrane fragments of ordered PSII arrays contain the

commonly found C2S2M2 structures of PSII. The work on membrane fragments was a major

advance towards understanding of PSII organization in the intact photosynthetic membrane.

The question arises to whether these fragments represent a typical organization of the

photosystem. This topic remained a subject of many debates for a number of years. Whilst

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the crystalline fragments of PSII are very useful for identification of the state of PSII in the

membrane it was argued that they are unlikely to represent a typical arrangement of this

photosystem in vivo . Indeed, thorough systematic investigations of freeze-fracture replicas

of photosynthetic membranes (Figure 7.1 ) reveal that the area of these structures occupies

not more than 10% of the total PSII space in the photosynthetic membrane. This value was

recently found to be dependent upon the composition of PSII light harvesting antenna and

will be discussed in detail in Chapter 9. One of the extreme examples of enhancement of

the proportion of the ordered PSII arrays was found in plants lacking the minor antenna

complex CP24. Figure 7.9 (c) shows the image of a PSII membrane fragment where the

C2S2 supercomplex structures form ordered arrays. The C2S2M2 structure is totally absent

in these plants. It is likely that CP24 indeed is responsible for the binding of the M LHCII

trimer to the PSII supercomplex. This minor antenna component is acting as the molecular

glue that ensures attachment of the second LHCII trimer to the supercomplex. Hence, the

earlier discussed idea that the minor PSII antenna can play a role of the linker for the major

LHCII to the core PSII complex seems to be reasonable. The work has simply shown that

CP24 has a specific function of M trimer binding to the supercomplex.

Remarkably, the inability of PSII particle to bind the second LHCII trimer resulted in the

formation of a large proportion of crystalline arrays of the photosystem (up to 30% of the total

PSII) suggesting that the peripheral LHCII antenna acts as an important breaker of the order in

the PSII-containing membrane domain. Therefore, LHCII seems to be the major factor that

defines the dynamic nature of the grana membrane. Indeed fluorescence recovery after pho-

tobleaching (FPAP) experiments on the membranes devoid of CP24 complex revealed that the

protein mobility there is almost two times slower compared to the wild type PSII membrane.

Hence, the ordered arrangement of PSII supercomplexes prevents membrane dynamics with a

likely effect on some of its functions. Naturally, the order here is counterproductive, since the

Figure 7.9 Fragment of the grana membrane containing ordered arrays of PSII (a). (b) Image of the ordered PSII structures produced using the single particle analysis approach. White arrows indicate the densities of LHCII trimers; the white dashed line highlights the contour of C2S2M2 particle. Reprinted with permission from Yakushevska et al . © 2003 American Chemical Society. (c) Ordered arrays of C2S2 particles of the membrane fragment from plants lacking CP24 minor antenna complex. Reproduced with permission from Kovacs et al . ( 2006 ).© 2011 American Society of Plant Biologists.

(a)

(b) (c)

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‘frozen’ membrane is likely to loose its vital functions. Therefore, breaking of order enables

the dynamics of crowded PSII membrane protein landscapes driven by the ambient tempera-

ture (in other words, thermodynamically-driven). Therefore, the nature using the heterogene-

ous multi-subunit structure of light harvesting antenna of PSII has a certain level of control

over the membrane fluidity and hence vital adaptive functions (see Chapter 9).

The next step in the investigation of the in vivo PSII organization was undertaken by

Boekema (2011) who applied cryoelectron microscopy to study several stacked grana mem-

branes. The advantage of cryoelectron microscopy is that it does not use any artificial envi-

ronment or staining procedures to fix the sample. Grana membranes have been isolated from

thylakoids using a gentle treatment with digitonin, a detergent that was used for isolation of

membrane fragments (see previously) and found reasonably mild and noninvasive. As a

result the whole intact grana membranes could be visualized albeit to a lower resolution of

4 nm only (Figure 7.10 a). Nevertheless, this enabled to identify densities of the dimeric PSII

Figure 7.10 (a) Cryoelectron microscopy image of the fragment of PSII membrane obtained from the tomographic reconstruction of grana particle embedded into ice at a 4 nm resolution (top). Bottom: enlarged area of the box (top) containing modelled C2S2M2 PSII particles. The star indicates one of the modelled C2S2M2 complexes. (b) Complementing the spaces between C2S2M2 complexes with LHCII trimers. Reprinted from Kouřil et al . © 2011 with permission from Elsevier.

(a)

(b)

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core complexes. They were fairly randomly redistributed in the membrane, confirming the

freeze-fracture microscopy data that found only small present of ordered PSII arrays.

Localization and orientation of the PSII dimeric cores was used as initial information to

model upon them the C2S2M2 supercomplex structures. The latter were simply overlaid and

aligned according to the PSII core orientations, as is shown in Figure 7.10 (a). The bottom

panel displays the enlarged membrane area highlighted on the EM image. Remarkably, PSII

complexes are not only randomly oriented but well-spaced from each other. The spaces

between the complexes are likely to be filled mostly with the rest of the LHCII: L-trimers.

Figure 7.10 (b) shows the result of modelling incorporation of the LHCII trimers into empty

spaces between C2S2M2 complexes. After the insertion of LHCII trimers the calculation of

the LHCII/RCII ratio yields approximately 5, which is consistent with the various biochem-

ical and structural evidence. Such organization of PSII should enable relatively efficient

connectivity between the units of photosystems and ensure the effective cross-section cor-

responding to the at least C2S2M2L6 organization of the PSII complex. However, it is fea-

sible to assume that only C2S2M2 structure is the stable ‘core’ of the complete PSII since it

is stabilized by the S and M trimer binding via the minor antenna complexes and the rest of

the trimers are relatively free to diffuse in the membrane (L-type). Hence, the central factor

in the stabilizing the structural heterogeneity of LHCII trimer binding to the PSII complex

is the minor LHCII antenna complexes CP24, CP26 and CP29.

The described cryoelectron microscopy approach is based upon the use of detergent that

still can cause a certain degree of the damage to the grana system and the membrane itself.

In some cases that concern physiologically-related phenomena in the whole of the thylakoid

membrane system the use of detergents is not welcome. For example, detergents can cause

uncoupling, that is, collapse of the proton gradient across the thylakoid membrane etc.

7.5 Robustness of the Light Harvesting Antenna Design: Resurrecting the Structure to Preserve the Function

A question of why the photosynthetic antenna has to be encoded by such relatively large

number of genes seems to be at least partially answered in the previous paragraph. Indeed,

structural work on PSII has revealed some heterogeneity within LHCII complexes. The

minor antenna is more intrinsic to the supercomplex and important in attaching S and M

LHCII trimers to the structure. The existence of the two major antenna pools, one attached to

the supercomplex and the other is loose ensures certain degree of membrane protein mobil-

ity, that is necessary for some adaptive functions. Another, and more striking phenomenon

related to the multi-subunit nature of light harvesting antenna was revealed using the Lhcb2

gene-silencing technique that greatly surprised researchers and highlighted the vital need for

LHCII antenna for PSII. Jansson used Lhcb2 antisense technique to inhibit the expression of

one of the major LHCII polypeptides that builds LHCII trimers, Lhcb2 (Andersson et al ., 2003). His group succeeded to eliminate almost totally Lhcb2 protein (Figure 7.11 ).

Surprisingly, the levels of another major LHCII polypeptide, Lhcb1, dropped to virtually

zero. Hence, they obtained plants lacking the two major polypeptides of trimeric light

Rupan_c07.indd 150 8/29/2012 5:36:54 PM


harvesting complex. In the wild-type plants this complex binds around 75% of the total PSII

chlorophyll, a truly major light harvesting antenna. One would expect plants suffer growing

under low light from the shortages of incoming light energy into the chloroplast membrane

and consequently grow much slower and be less productive (biomass, seeds). Another con-

sequence that was expected as a result of the absence of the major LHCII was lack of the

grana structure, since this complex was implied to be a major factor that is involved in the

grana formation (see Chapters 3 and 5 for details).

Growing the Lhcb2 antisense plants under low light environment (150 μ M m −2 s −1 ) enabled

researchers to reveal a number of surprising features. Whilst the total amount of chlorophyll

was reduced (only by ~30%) and plants looked paler than the wild type (Figure 7.11 ) plants

possessed normal photosynthetic capacity measured by CO 2 uptake and normal growth rate

under controlled light environment. In addition and more surprisingly, the antisense plants

showed grana in their chloroplasts (Figure 7.12 ). There was no alteration in the number of

grana or the number of thylakoids in grana stacks. Lhcb2 antisense plants seemed to render the

major LHCII a non-essential PSII component that is not important for the plant performance.

The apparent controversy prompted Ruban and colleagues (2003) to undertake a bio-

chemical and structural investigation of the photosynthetic membrane from Lhcb2 anti-

sense plants. The idea was to find out whether the membrane protein composition was

altered in the antisense plants. Protein analysis of PSII containing membranes revealed an

unusual phenomenon. Whilst Lhcb1 and 2 proteins were almost totally absent in the anti-

sense plants, Lhcb3 and Lhcb5, in particular, were overexpressed by 100 and 500% respec-

tively (Figure 7.13 ). The large increase in the level of CP26 complex could be expected in

Lhcb1

Wild

Wild type

as Lhcb2

Type

Lhcb2

Lhcb3

asLhcb 2

Figure 7.11 Arabidopsis plants lacking the major LHCII polypeptides, Lhcb1 and 2.

Rupan_c07.indd 151 8/29/2012 5:36:54 PM


this case. Therefore preparative biochemistry was a next step towards solving the puzzle of

the Lhcb2 antisense plants.

Isolated thylakoid membranes as well as PSII particles were prepared and subjected to

solubilization with n-dodecyl- α -D-maltoside, a mild detergent preserving macrostructural

organization of PSII (see previously). Both, thylakoids and PSII membranes from antisense

plants revealed the existence of bands corresponding to the elution time of LHCII trimers

(Figure 7.14 ). This fact, was again puzzling since according to the protein analysis the

major trimer-forming polypeptides were absent in these plants. The discovered ‘trimers’

were less stable than trimers of LHCII from the wild type. This was revealed by using

Lhcb

1Lhcb6

Lhcb5

Lhcb4

Lhcb3

Lhcb2

Lhcb1

PsbAWT As

0

100

200

300

Ban

d in

tens

ity (

% o

f wild

type

)

400

500

600

700

Lhcb

2

Lhcb

3

Lhcb

4

Lhcb

5

Lhcb

6

Figure 7.13 Western blot analysis of PSII membranes (BBY particles) of the wild type (wt) and Lhcb2 antisense Arabodipsis plants. Left: photo of the western blot; right: densitographic quantification of protein bands from the western blot. Reprinted by permission from Macmillan Publishers Ltd from Ruban et al . © 2005.

Figure 7.12 Presence of stacked grana thylakoids in Lhcb2 antisense plants.

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slightly higher DM concentration for solubilization, that yielded a much larger ‘monomer’

fraction and strongly depleted ‘trimer’ one (Figure 7.14 ). Therefore, it was concluded that

the trimers of Lhcb2 antisense plants can be broken into monomers even by using very mild

detergent. These trimer fractions were collected and subjected to the polypeptide analysis.

Western blots confirmed the absence of Lhcb1 and 2 polypeptides. Instead, the found trim-

ers were built of Lhcb5 polypeptide with the minor presence of Lhcb3 gene product

(Figure 7.14 ). Therefore, an important conclusion was reached as a result of these studies:

the trimeric structure of LHCII antenna was preserved despite the absence of the Lhcb1 and

2 polypeptides via their replacement by Lhcb5 polypeptide normally forming CP26 minor

monomeric antenna complex. Further analysis of this trimeric fraction revealed its hetero-

geneity. Two types of trimers were prepared using nondenaturing IEF (see Chapter 4): one

type was a homotrimer built exclusively of Lhcb5 polypeptides and the other was a hetero-

trimer built of Lhcb5 and Lhcb3 polypeptides. The latter was found to be more stable than

the former.

Further work on solubilized thylakoid membranes revealed yet another phenomenon: the

presence of loosely associated LHCI complexes with PSI that can be relatively easily

removed from the photosystem by nonionic detergent. Normally, as shown previously, LHCI

can only be removed from PSI core using multi-ionic detergents. Here, it was clearly not the

case. Estimations of the PSI antenna size suggested that the loosely-bound LHCI complexes

0.015 20 25

Retention time, min

30 35

0.5

1.0

Abs

orpt

ion

at 6

76 n

m, r

el.

1.5

2.0

2.5

High DM

as

wt

PSIIs

PSI

PSIIc

PSITrimer

Monomer

LHCII

Lhcb5

Lhcb3

Lhcb2

Lhcb1wt as

Figure 7.14 Gel filtration FPLC chromatograms of solubilized PSII particles with 0.85% (Low DM) and 1.2% (High DM) n-dodecyl α -D-maltoside from wild type (wt) and Lhcb2 ant ise nse (as) Arabidopsis plants. PSIIs and c are PSII supercomplexes and core complexes, respectively. Vertical lines indicate run times of LHCII trimers and monomers, respectively. Bottom runs are of sucrose gradient centrifugation isolated PSI and LHCII complexes. Encircled is a discovered trimeric LHCII band in the antisense plants. Inset: western blots of the ‘trimer’ fractions from the wild-type (wt) and antisense (as) plants.

Rupan_c07.indd 153 8/29/2012 5:36:55 PM


are likely to be ‘extra’ light harvesting units. This could explain their ‘loose’ binding, since

according to the PSI structure (Figure 7.8 ) these extra antenna complexes should interact

with already bound LHCI complexes or the PSI sites that have much lower affinity of LHCI

binding than the original ones. Interestingly, that it was reported that PSI from the green

algae Chlamidomonas contained up to six extra LHCI units in comparison to the higher plant

PSI. It is possible to imagine, that at certain environmental conditions the higher plant PSI

does regulate its LHC antenna size by forming larger and more loosely-bound LHCI com-

plexes. In fact, they can be defined as peripheral LHCI complexes similarly to L-trimers of

PSII, that constitute more then 50% of the whole of LHCII antenna. This consideration draws

an interesting similarity between the light harvesting antenna design of PSI and PSII, a gradi-

ent in LHC antenna binding affinity to photosystem macrostructure. The physiological sense

for such design can be found in ability to effectively regulate the cross-section according to

the environmental conditions (see Chapter 9).

The ultimate evidence that Lhcb2 antisense plants possess trimeric light harvesting com-

plexes came from the structural work based upon electron microscopy combined with the

single particle analysis approaches. Figure 7.15 displays the results of this work. The

C2S2M2 structure was found to be the dominating state of the PSII supercomplex with two

LHCII trimers attached to the dimeric core along with the minor antenna. This structure is

Figure 7.15 The structure of PSII C2S2M2 particle from the Lhcb2 antisense plants (top). Bottom: arrangement of LHCII trimers from the wild-type (wt) and antisense (as Lhcb2) plants in the crystalline arrays of PSII. Reprinted by permission from Macmillan Publishers Ltd Ruban et al . © 2003

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virtually undistinguishable from the structure of C2S2M2 complex from the wild-type.

Hence, the overexpression of Lhcb5 complex made it possible not only to build trimeric

LHCII but also to preserve the structure of the PSII supercomplex. This was apparently a

crucial step towards the preservation of the grana structure and ensured the high photosyn-

thetic performance of the antisense plants described previously. Figure 7.15 also shows the

structure of interacting trimers of C2S2M2 complexes within the crystallinity arrays found

in the mutant plants. The packing of the antisense LHCII trimers seems to be slightly altered

in comparison to the wild-type ones, suggesting the subtle changes in their geometry most

likely due to the presence of Lhcb5 proteins instead of Lhcb1 and 2. Hence, a hint of not

entirely exact organization, that does apparently not alter significantly the PSII performance.

The existence of trimers in the antisense plants suggests a very special requirement for the

LHCII trimeric structure. This could be both functional (in light harvesting) and structural

(in assembly of the PSII macrostructure). The trimerization of LHCII appears to create fea-

tures that are important for the promotion of efficient energy transfer. The cluster consisting

of Chl a 610-612 may be the channel for energy transfer to neighbouring antenna and core

complexes, and the presence of three identical clusters on each trimer could enable efficient

energy transfer within the PSII macrostructure, bearing some similarity to the pigment rings

found in bacterial antenna complexes (see Chapter 6). Figure 7.16 summarizes the structure

of the PSII supercomplex from the Lhcb2 antisense plants. Whilst the core and minor

antenna remain unaffected, the S and M trimers are built of Lhcb3 and, mainly, of Lhcb5

polypeptides. The M and S timers, which are part of the supercomplex, form an intrinsic part

CP26

CP43

CP43

CP47CP47

CP29

CP26

CP29

CP24

D2

D2

D1

D1

Lhcb

3

Lhcb5

Lhcb5

Lhcb

5 Lhcb5

Lhcb

5 Lhcb5

Lhcb3

Lhcb5

LHCIIb

Figure 7.16 Schematic presentation of the organization of PSII supercomplexes from plants lacking the major LHCII polypeptides. D1,2 are reaction centre polypeptides; CP43,47 are the core antenna complexes; CP24,26,29 are the minor LHC antenna complexes; Lhcb3 and 5 are the polypeptides that form the peripheral trimeric antenna complexes.

Rupan_c07.indd 155 8/29/2012 5:36:56 PM


of the macro-organization of PSII in the granal membranes, whereas the remaining trimers

appear to be located in PSII-free membrane regions. In the wild type plants the M trimer

consists of Lhcb1 and Lhcb2, whereas the S trimer is composed of Lhcb1 and Lhcb3. It was

proposed that in as Lhcb2 plants M trimer is a homotrimer of Lhcb5 and S trimer is a het-

erotrimer of Lhcb3 and Lhcb5 polypeptides. The modified plants revealed a specific response

of the light harvesting gene family and photosystem assembly mechanisms to the absence of

the major LHCII polypeptides. The most related minor antenna protein to the Lhcb1-3 poly-

peptides, Lhcb5, possessing a specific trimerization motif (see previously) was overex-

pressed in plants missing Lhcb1 and 2 proteins. The overexpression led to the assembly

system to recognize Lhcb5 as a building block of LHCII major antenna trimeric state. The

complexes were not only assembled but even incorporated in the stoichiometric amounts

into the PSII supercomplex structure, typical of the wild-type plants. The original functions

of the major LHCII; grana stacking and efficient light harvesting have been restored. One

single protein expression levels made the whole difference for the fate of plant development,

photosynthetic performance and survival. Such compensatory phenomenon was called

robustness of light harvesting antenna design. It highlights the sheer importance of the light

harvesting antenna in plants and constitutes one of its central properties.

This chapter aimed to demonstrate the interconnection between the composition of pho-

tosystem antenna complexes and their assembly into the macrostructures and eventually

into the membrane that is ‘packed’ with proteins. The multi-subunit structure of light har-

vesting antenna makes its functions flexible and robust. The antisense work described here

demonstrated the vital fundamental need in the light harvesting antenna for the efficient

photosynthetic performance essential for plant life. Apparently the primary structure of

proteins involved is not only a major determinant of the secondary and tertiary structure but

also the quaternary structure of photosystem complexes and dynamics of the whole photo-

synthetic membrane.

References

Andersson , J. , Walters , R.G. , Horton , P. and Jansson , S. ( 2001 ) Antisense inhibition of the photosyn-thetic antenna proteins CP29 and CP26: implications for the mechanism of protective energy dissipation . Plant Cell , 13 , 1193 – 1204 .

Andersson , J. , Wentworth , M. , Walters , R.G , et al . ( 2003 ) Absence of Lhcb1 and Lhcb2 proteins of the light-harvesting complex of photosystem II- effects on photosynthesis, grana stacking and fitness . The Plant Journal , 35 , 350 – 361 .

Bassi , R. , Høyer-Hansen , G. , Barbato , R. , et al . ( 1987 ) Chlorophyll-proteins of the photosystem II antenna system . J. Biol. Chem. , 27 , 13333 – 13341 .

Ben-Shem , A. , Frolow , F. and Nelson , N. ( 2003 ) Crystal structure of plant photosystem I . Nature , 426 , 630 – 635 .

Boekema , E.J. , Hankamer , B. , Bald , D. , et al . ( 1995 ) Supramolecular structure of the photosystem II complex from green plants and cyanobacteria . Proc. Natl. Acad. Sci. U. S. A. , 92 , 175 – 179 .

Boekema , E.J. , van Roon , H. , van Breemen , J.F.L. and Dekker , J.P. ( 1999 ) Supramolecular organiza-tion of photosystem II and its light-harvesting antenna in partially solubilized photosystem II membranes . Eur. J. Biochem. , 266 , 444 – 452 .

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Dekker , J.P. and Boekema , E.J. ( 2005 ) Supramolecular organization of thylakoid membrane proteins in green plants . Biochim. Biophys. Acta , 1706 , 12 – 39 .

Fradkin , L.I. , Kolyago , V.M. and Shlyk , A.A. ( 1972 ) Gel-electrophoretic fractionation of the digi-tonin-treated barley chloroplasts . Dok. l Akad. Nauk. SSSR , 207 , 453 – 456 .

Jansson , S. ( 1999 ) A guide to the Lhc genes and their relatives in Arabidopsis . Trends in Plant Sci. , 4 , 236 – 240 .

Jansson , S. ( 1994 ) The light-harvesting chlorophyll a/b binding-proteins . Biochim. Biophys. Acta , 1184 , 1 – 19 .

Johnson , M.P . Goral , T.K ., Duffy , C.D.P ., et al . ( 2011 ) Photoprotective energy dissipation involves the reorganization of photosystem II light harvesting complexes in the grana membranes of higher plant chloroplasts . Plant Cell , 23 , 1468 – 1479 , www.plantcell.org American Society of Plant Biologists .

Kouřil , R. , Dekker , J.P. and Boekema , E.J ( 2012 ) Supramolecular Structure of photosystem II in green plants . Biochim. Biophys. Acta , 1817 , 2 – 12 .

Kouřil , R. , Oostergetel , G.T. and Boekema , E.J. ( 2011 ) Fine structure of granal thylakoid membrane organization using cryoelectron tomography . Biochim. Biophys. Acta , 1807 , 368 – 373 .

Ruban , A.V , Wentworth , M. , Yakushevska , A.E. , et al . ( 2003 ) Plants lacking the main light harvesting complex retain photosystem II macro-organisation . Nature , 421 , 648 – 652 .

Staehelin , L.A. ( 2003 ) Chloroplast structure: from chlorophyll granules to supra-molecular architec-ture of thylakoid membranes . Photosynthesis Research , 76 , 185 – 196

Umena , Y. , Kawajkami , K. , Shen , J.-R. and Kamiya , N . ( 2011 ) Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9Å . Nature , 473 , 55 – 60 .

Yakushevska , A.E. , Keegstra , W. , Boekema , E.J. , et al . ( 2003 ) The structure of photosystem II in Arabidopsis : localization of the CP26 and CP29 antenna complexes . Biochemistry , 42 , 608 – 613 .

Bibliography

Andersson , J. , Wentworth , M. , Walters , R.G. et al . ( 2003 ) Absence of Lhcb1 and Lhcb2 proteins of the light-harvesting complex of photosystem II- effects on photosynthesis, grana stacking and fitness . The Plant Journal , 35 , 350 – 361 .

Ballottari , M. , Girardon , J. , Dall ’ Osto , L. and Bassi , R. ( 2012 ) Evolution and functional properties of Photosystem II light harvesting complexes in eukaryotes . Biochim. Biophys. Acta , 1817 , 143 – 157 .

Bassi , R. , Sandona , D. and Croce , R. ( 1997 ) Novel aspects of chlorophyll a/b -binding proteins . Physiol. Plantarum , 100 , 769 – 779 .

Boekema , E.J. , Hankamer , B. and Barber , J. ( 1997 ) Structure and membrane organisation of photo-system II in green plants . Annu. Rev. Plant Physiol. Plant Mol. Biol. , 48 , 641 – 671 .

Camm , E.L. and Green , B.R. ( 2004 ) How the chlorophyll-proteins got their names . Photosynth. Research , 80 , 189 – 196 .

Fradkin , L.I. , Chkanikova , R.A. and Shlyk , A.A. ( 1981 ) Coupling of chlorophyll metabolism with submembrane chloroplast particles, isolated with digitonin and gel electrophoresis . Plant Physiol. , 67 , 555 – 559 .

Green , B.R. and Durnford , D.G. ( 1996 ) The chlorophyll-carotenoid proteins of oxygenic photosyn-thesis . Annu. Rev. Plant Physiol. Plant Mol. Biol. , 47 , 685 – 714 .

Kovacs , L. , Damkjær , J. , Kereïche , S. , et al . ( 2006 ) Lack of the light-harvesting complex CP24 affects the structure and function of the grana membranes of higher plant chloroplasts . Plant Cell , 18 , 3106 – 3120 .

Pichersky , E. and Green , B.R. ( 1990 ) The extended family of chlorophyll a/b -binding proteins of PSI and PSII , in: Proceedings of the VIIIth International Conference on Photosynthesis Stockholm,

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Sweden, August 6–11, 1989. Current Research in Photosynthesis , M. Baltschffsky (ed.), Vol. 1, pp. 53–556. Kluwer.

Rigoni , F. , Barbato , R. , Friso , G. and Giacometti , G.M. ( 1992 ) Evidence for direct interaction between the chlorophyll-proteins CP29 and CP47 in Photosystem II . Biochem. Biophys. Res. Commun. , 184 , 1094 – 1100 .

van Roon , H ., van Breemen , J.F.L ., deWeerd, F.L. et al . ( 2000 ) Solubilization of green plant thyla-koid membranes with n-dodecyl- α ,D-maltoside. Implications for the structural organization of the Photosystem II, Photosystem I, ATP synthase and cytochrome b(6)f complexes . Photosynth. Res ., 64 , 155 – 166 .

Ruban , A.V. , Solovieva , S. , Lee , P.J. , et al . ( 2006 ) Plasticity in the composition of the light harvesting antenna of higher plants preserves structural integrity and biological function . J. Biol. Chem. , 281 , 14981 – 14990 .

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‘If you want to make a song more hummy, add a few tiddely poms ...’ Winnie-the-Pooh (by Alan Alexander Milne)

In a broader sense, spectroscopy is an analysis based on the interaction between electro-

magnetic radiation and matter as a function of wavelength or frequency. Several spectro-

scopic techniques used for studies of membrane proteins and the photosynthetic light

harvesting antennae in particular, are already described in Chapter 4. Functions of majority

of proteins in nature are not based upon interaction with light; nevertheless, many of them

can also be studied by spectroscopy, though mainly in UV and IR regions. The photosyn-

thetic antenna is the most adequate object for the spectroscopic studies by nature since its

function is actually based upon interaction with light radiation. The pigments of light har-

vesting antenna conceal great deal of important information about their energies, excited

state properties, conformation, orientation, interactions with proteins and each other as

well as the other features that are vital for the antenna function. In addition, some spectro-

scopic methods could give information of a structural character about pigments and the

protein itself. This information can be invaluable in making correct assignments of the

structural elements of protein as well as for understanding how protein works. Hence, it is

not surprising that the literature on spectroscopic research of the photosynthetic membrane

is vast counting literally thousands of papers. Light spectroscopy is a great and, in many

cases, noninvasive tool that can be applied for the studies of isolated complexes, mem-

branes, chloroplasts and even intact leaves. The other major advantage of many spectro-

scopic techniques is selectivity . Indeed, some spectroscopic methods can be applied to very

complex samples, containing thousands of different types of molecules, many of them

Dynamics of Light Harvesting Antenna: Spectroscopic Insights

8

Rupan_c08.indd 159 8/29/2012 5:35:59 PM


optically active in the visible region. Yet, these approaches can specifically identify and

monitor properties of particular types of molecules even if they are very minor components

of the system under investigation. Therefore, not only are spectroscopic methods good for

identification and evaluation of amounts of specific substances in complex samples, they

are also useful in the studies of molecular dynamics that is often connected to the molecu-

lar mechanisms of a variety of biological processes.

Spectroscopic approaches used in studies of the photosynthetic membrane can arbitrar-

ily be divided into the two major classes: passive and active . Passive methods do not alter

the state of the sample during the spectral measurements; whilst active use a number of

factors that alter the sample. Both groups of approaches aim at a certain level of selectivity

or specificity, since the photosynthetic membrane or even the isolated antenna complexes

are very heterogeneous systems, containing variety of pigments. The active group of spec-

tral approaches aims in the majority of cases to enhance selectivity and insightfulness,

gaining more specific and, at the same time, more profound information about the state of

chromophores under investigation. One of the examples of active type of spectroscopy is

light-induced approaches. They can be steady-state, when sample is being measured during

relatively long time (minutes or longer) after or during illumination or time-resolved, when

the fast sample response to light in non-equilibrium state is being recorded. The latter

approach is called transient spectroscopy. The time resolution of this method can vary in

the time scale from seconds to femtoseconds. Other examples of active type spectroscopy

are temperature-, pressure-, solvent-, field (electric or magnetic) induced spectroscopies. In

fact, these are all differential methods that utilize the principle of alteration of a relatively

small population of spectral states or components in the sample, hence gaining selectivity.

8.1 Steady-State Optical Spectroscopy of LHCII : Composition and Order

Among passive spectroscopic methods are absorption, fluorescence, linear and circular

dichroism and many other spectroscopies that simply measure spectra without using any

sample-altering factors during or before the measurements: the sample state remains largely

constant. Figure 8.1 represents the three different types of spectroscopies applied to study

LHCII antenna complex. Absorption spectrum of isolated LHCII contains many bands

reflecting various electronic transitions from the ground to the excited state. This type of

spectroscopy, therefore, gives first of all information on the sample composition. The variety

of transitions originates from the presence of eight chlorophyll a , six chlorophyll b and four

xanthophyll molecules. Absorption of each is represented by several electronic transitions.

Chlorophylls absorb in the blue (Soret) and red regions. Carotenoids absorb in the blue and

possess at least three clearly defined bands (see Chapter 5 for more details). In order to esti-

mate number and maxima positions of absorption spectral components a second derivative

calculation can be applied (see Chapter 4). This simple procedure produces significantly

better-resolved absorption spectrum. It can be seen from the Figure 8.1 (a) that there are at

least five bands in the red region of chlorophyll absorption at this temperature (77 K). The

Rupan_c08.indd 160 8/29/2012 5:36:00 PM

Dynamics of Light Harvesting Antenna: Spectroscopic Insights 161

450

–0.1

–0.05

0

Circ

ular

dis

chro

ism

, rel

. 0.05

–0.1

0

0.1

Line

ar d

ichr

oism

, rel

. 0.2

0

0.1

0.2

0.3

Abs

orpt

ion

0.4

0.5

500 550Wavelength, nm

600 650 700

472

490

438

510

500450

448472

485510 642

676 700650600550500

x(–125)

457415 510 638

Composition

495

485

472440

Chl a Chl b Xanthophylls Chl b Chl a

433

450

650

Order

550 600 650 700

457

640650 678

Order

650

670

676

661

+ =

+ =

(a)

(b)

(c)

Figure 8.1 Absorption (a), Linear dichroism (b) and circular dichroism (c) spectra of isolated trimeric LHCII complex. Absorption and Linear dicroism spectra were recorded at 77K. Circular dichroism spectrum was recorded at room temperature.

Rupan_c08.indd 161 8/29/2012 5:36:00 PM


lower the temperature, the better is the spectral resolution since the bandwidth become pro-

gressively narrower with the temperature fall. Clearly number of chlorophyll bands here

does not much either the number of chlorophylls (14) or number of chlorophyll types (two,

a and b ). It is somewhat in between, meaning that there is clear variety of chlorophyll a or b

forms originating from the environmental effects within the LHCII complex, hence the

number of bands is higher than two. However, some chlorophylls can have similar absorp-

tion maxima, hence the number of transitions observed is less than 14. It also may well be

possible that not all spectral components have been resolved by the second derivative

approach due to a large spectra overlap and relatively broad bandwidth. The Soret band

region reveals even more complex structure compared to the red band. Around 10 bands and

shoulders could be resolved by the derivative method. Group of bands at 440 nm and a band

at 470 nm belong to chlorophyll a and b , respectively. Absorption of xanthophylls clearly

overlaps with chlorophyll b band and stretches up to 510 nm. Assignment of xanthophyll

transitions has always been a big challenge. Selective spectroscopic approaches are required

for this purpose that will be described in Section 8.4 of this chapter.

The linear dichroism spectrum (LD) of LHCII is shown in Figure 8.1 (b). As was described

in Chapter 4 the method uses oriented sample (in the stretched gel) and differential absorp-

tion of light polarized in two planes, perpendicular and parallel to the plane of the sample

orientation. This method provides clues on the order of pigments in LHCII since it reflects

the orientation of their transition dipole moments. The spectral components are better

resolved in this spectrum compared to absorption one. This is largely due to the differential

character of the measurement as well as the existence of pigments (groups of pigments) with

preferential orientations, a sign of order. In LHCII the red-most chlorophyll a ( a 610–612)

(the terminal emitter) transition dipole moment is oriented approximately in parallel to the

particle (membrane) plane (Figure 8.2 ). Therefore, the amplitude of the red-most band in

LD spectrum is dominating (676 nm positive band). Apart from the orientation of individual

a610

a612a611

Figure 8.2 Structure of the LHCII monomer displaying the three chlorophyll a molecules of the terminal emitter, a610, 611 and 612. Short black arrows show the orientations of the chlorophylls’ transition dipole moments. Long grey arrow indicates the orientation of the membrane plane.

Rupan_c08.indd 162 8/29/2012 5:36:01 PM


pigments the LD spectrum can be affected by transitions originated from pigment-pigment

interactions of excitonic character. The two splitting components can have opposite orienta-

tions of their transition dipoles. This feature can often undermine the amplitude of LD spec-

trum and cause the appearance of spectral components that is difficult to relate to the

structure of the absorption spectrum. The Soret region of the LD spectrum of LHCII dis-

plays a well-resolved structure with four clearly visible bands at 448, 472, 485 and 510 nm.

The three last transitions have their analogues in the absorption spectrum. This suggests that

the transition dipole moments of the pigments responsible for them have similar orientation.

The third spectrum shown in the Figure 8.1 (c) is the circular dichroism spectrum (CD).

This spectroscopy does not require sample orientation and provides useful information

even if measured at room temperature. Differential absorption of the circularly polarized

light is a useful probe of the pigment structural configuration and pigment-pigment interac-

tions. Bands registered in CD spectra can be of different molecular nature. For the simple

isolated molecule CD band arises from intrinsic asymmetry of a molecule. In this case one

electronic transition will show a CD band of the same shape as the absorption. The sign

(‘+’ or ‘−’) will depend upon the handedness of the molecule. When pigments interact to

form dimers or aggregates, CD will be originated from the excitonic coupling between

them. Excitonic interactions give rise to the positive and negative bands of the spectrum.

The CD spectroscopy is another complimentary method to absorption methods reflecting

the order in the LHCII complex. Correct binding of pigments in their pockets, interactions

with the other pigments of the complex (see Chapter 6 for examples) can be detected and

monitored by the CD spectroscopy. This method is very useful in the LHCII reconstitution

work (see Chapter 4) as one of the major tests for the correct assembly of the complex. In

case if the latter is not correctly assembled the CD spectrum will significantly change with

sometimes complete disappearance of structure and amplitude of bands. The CD spectros-

copy is also a prompt and sensitive tool to analyse the oligomerization state of LHCII.

When the complex monomers assemble into the trimeric state a distinctive negative shoul-

der at about 640 nm will emerge along the 650 nm band of chlorophyll b (Figure 8.1 c). In

addition the negative band in the Soret region at 490 nm will broaden revealing the shoul-

der at 510 nm. The relative strength of 678 nm negative band reflects the state of the termi-

nal emitter chlorophylls a 610–612. Their interactions and configuration is important to

ensure their function, efficient reception of excitation energy from all chlorophylls as well

as relatively long excited state lifetime.

Steady state fluorescence spectroscopy of the photosynthetic membrane components is

another widely used tool. As was shown in Chapter 5 it can be used on variety of systems

from isolated complexes to intact chloroplast membranes. This method reflects energy

transfer between antenna pigments, hence can be used for testing the intactness of recon-

stituted antenna complexes in vitro along with the CD spectroscopy. Figure 8.3 displays

77 K fluorescence spectra of intact and denatured LHCII complex. The spectrum of intact

complex is represented by one narrow chlorophyll a band peaking at 680 nm and a struc-

tured vibronic satellite (see Chapter 5). The spectrum of the denatured complex is very

different. It is much broader and contains a strong shoulder of chlorophyll b fluorescing at

~655 nm. The presence of this shoulder indicates poor energy transfer from chlorophyll b

Rupan_c08.indd 163 8/29/2012 5:36:01 PM


to chlorophyll a , hence lack of required order in the ‘programmed solvent’ of LHCII. The

main band broadening is caused by the exposure of chlorophyll a to a variety of microen-

vironments, an inevitable result of the entropy increase and loss of order. The order of the

native LHCII is manifesting from the uniform reproducible arrangement of pigments in

very similar environment of each of the trimer/monomer. When the complexes deteriorate

with the temperature increase (as in Figure 8.3 ), they have more freedom to adopt a multi-

tude of conformations that determine the environment of pigments. These variations in

microenvironment cause variations in the absorption maxima positions that ultimately

cause the integral fluorescence spectrum broadening. Apart from the entropic effect the

broadening is also due to the fact of the energy transfer loss between pigments as in the

case of chlorophyll b (655 nm band) and some short wavelength chlorophyll a fluorescing

around 665–670 nm and contributing to the integral spectrum broadening.

The denaturing experiment indicates that the LHCII protein secondary and tertiary

structures are essential for the correct assembly of pigments into a coherently functioning

system fulfilling the light harvesting antenna function, efficient light energy collection and

transfer towards the terminal emitter chlorophyll. In the denatured LHCII the energy trans-

fer is clearly impaired, hence the light harvesting function is undermined.

The described steady-state spectroscopic methods provide fundamental information that

can be used for developing models of energy transfer between pigments of antenna. These

models use structural data and quantum mechanical simulations of spectra with parameters

taken from the band positions, amplitudes and band widths to assess interaction energies

640 660 680

Intact

Chl b

Denatured

LHCII

0.0

0.5

1.0

Flu

ores

cenc

e, r

el.

1.5

700Wavelength, nm

720 740 760

Figure 8.3 77 K fluorescence spectra of isolated intact (solid line) and denatured (dashed line) LHCII. Vertical short arrow indicates the chlorophyll b fluorescence shoulder. Fluorescence was excited at 435 nm.

Rupan_c08.indd 164 8/29/2012 5:36:01 PM


between pigments and directionality of the energy transfer within the whole light harvest-

ing complex. This is an ambitious task and the modelling work of this kind remains largely

in progress reflecting the sheer complexity of electronic energy landscapes in light harvest-

ing pigment-protein complexes.

8.2 Time-Resolved Spectroscopy of LHCII : Energy Migration

8.2.1 Time-Resolved Fluorescence Spectroscopy

Spectroscopy with time resolution or time-resolved spectroscopy is an attractive incisive

pool of methods that asses the dynamic behaviour of excitation energy in the photosyn-

thetic pigments, identifying its migration pathways and, in general, the efficiency of

light harvesting antenna. As was described in Chapter 5, one of the major antenna

parameters is the excitation energy lifetime, the time excitation lives in antenna. It is

determined by the excited state lifetime of individual pigments, energy transfer between

them (connectivity) and the presence of dissipative energy traps that can receive energy

from pigments and relatively quickly dissipate it into heat. Chlorophyll fluorescence is

a useful signal that reflects the excited state lifetime (Chapter 4, Eq. 4.8) and can be

relatively easily measured and quantified. Figure 8.4 (a) shows typical fluorescence

decay profile of isolated LHCII trimers. It is basically a redistribution of photons emit-

ted by the sample from the moment of laser light absorption. The laser pulse has to be

much shorter than the fluorescence decay to enable the reasonably high time resolution.

Logarithmic conversion of the fluorescence photon counts gives ideally a linear func-

tion of time (Chapter 4, Eq. 4.9) in case of monoexponential decay, as is shown in the

Figure 8.4 (b). Indeed, the fluorescence decay here is practically monoexponential. The

tangent of the decay trace corresponds to the sum of all radiative and nonradiative decay

rate constants, ( k f + k

nr ). The measured fluorescence lifetime will be reciprocal to this

sum ( τ f = 1/ (k

f + k

nr )). Hence, the experimentally determined fluorescence lifetime con-

tains important information about the nonradioactive fate of the excitation energy in

antenna. In the case of the isolated LHCII trimer the decay is simple and reflects the

lifetime of excitation of the terminal emitting chlorophylls a 610–612. Since the tempo-

ral resolution in the Figure 8.4 fluorescence traces is only ~50 ps, processes of energy

transfer within LHCII pigments that take about 1–2 ps to reach the terminal emitter

cannot be detected. All that is registered is the final stage of energy stabilization on the

terminal emitter that presents the collected excitation energy concentrated on the few

red-most chlorophylls. The fluorescence lifetime is found to be ~4 ns, more than three

orders of magnitude longer than it takes to reach the terminal emitter from the moment

of absorption. Hence, we see the fact of slowing down and storing the energy of light in

light harvesting antenna and making it available for the transfer to the reaction centre.

If the population of isolated LHCII trimers becomes heterogeneous for some reason

(sample denaturation, etc.), then the fluorescence decay becomes more complex and requires

careful analysis in order to determine the number of components and their lifetimes. When

Rupan_c08.indd 165 8/29/2012 5:36:01 PM

2000

Laser pulse

Laser pulse

Fluorescence decay

Fluorescence decay

Linear

Logarithmic

4000

6000

8000

10000

1

10

6 8 10 12 14

Time, ns

16

–4

16000

14000

12000

10000

8000

Flu

ores

cenc

e am

plitu

de, r

el.

6000

4000

2000

0660 680 700

Wavelength, nm

720 740 760

–2024

100

1000

10000

Flu

ores

cenc

e, p

hoto

nsF

luor

esce

nce,

pho

tons

PSII~2.0 ns

~100 psPSI

(a)

(b)

(c)

Figure 8.4 Chlorophyll a fluorescence decay of isolated trimeric LHCII represented in linear (a) and logarithmic (b) scales. Bottom panel in (b) is the difference between experimental and theoretically fitted decay traces. The laser pulse wavelength was 472 nm with decay time of ~70 ps. Fluorescence was detected at 680 nm. (c) Decay-associated spectra of PSI and PSII in isolated chloroplasts. All measurements are taken at room temperature.

Rupan_c08.indd 166 8/29/2012 5:36:01 PM


the heterogeneity of the sample is caused by the presence of spectrally distinct emitters, that

is, the presence of the blue-shifted chlorophyll of denatured complexes or the presence of a

mixture of antenna complexes with different spectral properties (LHCII, CP43, CP47, PSI,

etc.), that indeed takes place in the intact photosynthetic membrane, it is often useful to

apply the method of the global analysis. Global analysis allows calculation of the decay-

associated spectra (DAS) to see the fluorescence spectral profiles of different lifetime com-

ponents. Figure 8.4 (c) shows a result of a global analysis done on the isolated chloroplast

membranes at room temperature at the conditions of all closed PSII reaction centres (F m , see

Chapter 4). Here the two major distinct spectral and temporal components have been iso-

lated. One component with ~2 nm fluorescence lifetime belongs to the whole of PSII

antenna and the other component of ~100 ps lifetime originates from PSI. Indeed, isolated

PSI has a steady-state fluorescence spectrum that is very similar to the DAS of PSI. The

DAS of PSII, on the other hand, resembles closely the fluorescence spectrum of isolated

PSII membranes. Therefore, the global analysis of the fluorescence decay allows derivation

of spectral features of photosystems without their isolation. Hence, it is nondestructive and

a very informative approach. Lowering the temperature of experiment to 77 K (liquid nitro-

gen) strongly changes fluorescence spectra of photosystems (see Chapter 5, Figure 5.19).

Photosystem I spectrum increases in intensity and strongly red-shifts towards 733 nm.

Photosystem II gains an extra distinct band at 695 nm. DAS spectra taken at the low tem-

perature become more complex carrying information about a number of newly emerged

bands and energy transfer relations between the complexes of photosystems.

The results of time-resolved fluorescence experiments can be useful for the modelling

of the energy migration between various complexes like the PSII supercomplex. Figure 8.5

shows the real topography of the complex, taken from the single particle work described

in Chapter 7, with indications of the possible energy migration pathways between its

components. The modelling of this migration process does not take into account details of

the energy transfer between individual pigments within each complex but simply treats

the process as the excitation hopping between the PSII antenna and reaction centre com-

ponents. Therefore, it is called a coarse-grained model that treats energy migration as a

random walk process within the photosystem superstructure. Naturally, the application of

this approach became possible only relatively recently, after the single particle electron

microscopy has advanced our knowledge about the topology of the photosynthetic com-

plexes in vivo . The model takes into account the number of pigments associated with

different components. It allows determining the excitation hopping time between the

complexes and the trapping time by the reaction centre, both contributing to the average

fluorescence lifetime. Hence, it became possible to estimate the character of light harvest-

ing processes in photosystems defining the type of the photosystem design, migration-

limited (slow energy migration in antenna and fast trapping) or trap-limited (fast energy

migration in antenna and slow trapping).

8.2.2 Time-Resolved Absorption Spectroscopy

Time-resolved absorption measurements have been used for a long time in photosynthe-

sis research. Light-induced changes in absorption spectrum of chloroplasts or even leaves

Rupan_c08.indd 167 8/29/2012 5:36:02 PM


could provide information about various stages of charge separation and energy transfer

in the photosynthetic electron transport chain. In addition, absorption changes due to

electric field effect generated by the charge separation in PSII called electrochromic shift

(ECS) have been monitored. The absorption changes connected to the electron/proton

transport activity are relatively slow, occurring on the timescale of micro and millisec-

onds (see Figure 1.1), hence do not require very sophisticated equipment to be registered.

Events that involve the processes of charge separation in reaction centres and energy

transfer in antennae occur on much faster scale of pico- and even femtoseconds (Figure 1.1

7

19

21

5

2018

17

9

16CP29

14

22 CP2415

13CP47

D1/D2

0

CP4323

CP26

22

16

15

D1/D2

12

CP47 1

CP29

CP243 10

42

68

CP4311

CP26

103

4

9

17

7

19

5

21

Figure 8.5 Modelling excitation energy transfer between different complexes of the PSII C2S2M2 supercomplex. Numbering of the complexes is used for identification of excitation pathways (thick bars). Reproduced from Valkunas et al . with permission of the PCCP Owner Societies.© 2009 The Royal Society of Chemistry/PCCP.

Rupan_c08.indd 168 8/29/2012 5:36:02 PM


of Chapter 1). Processes that occur within the single photosynthetic antenna complex,

photon capture and energy transfer between individual pigments take place on the subpi-

cosecond timescale and therefore cannot be easily measured using the time-resolved

fluorescence technique described previously. Monitoring the changes in the absorption

spectrum following excitation within the timescale of femtoseconds can give direct infor-

mation about the evolution of excited states of the pigments of the antenna complex.

Performed with spectral resolution this approach can produce transient absorption spec-

tra, a range of temporal cross-sections that tracks the fate of excited states. Figure 8.6

shows the spectrum of absorption changes in isolated LHCII trimers following illumina-

tion with 100 fs pulse at the main red chlorophyll band absorption. The pump light was

of intensity high enough to prompt a relatively large population of chlorophyll a into

excited, S 1 , state. This caused disappearance of a large number of ground states, S

0 , hence

inability of the sample to absorb light photons with wavelength/energy of around S 0 -S

1 as

well as S 0 -S

2 energies. This manifested in the loss of absorption even after 300 fs of the

pump light application at ~675 and 440 nm regions as measured by the white probe light

pulse. The large bleach in the S 0 -S

1 transition region gradually disappears (vertical up

arrow) with relaxation of the first excited state into S 0 . The rate of this process reflects the

chlorophyll a excited state (S 1 ) lifetime. The broad structured positive absorption around

Figure 8.6 Light-induced changes in absorption spectrum of LHCII trimer. The difference spectrum (transient absorption) was measured 300 fs after sample illumination with the 100 fs pump pulse at 675 nm. The grey box highlights the region where xanthophyll S 1 -S n transition could take place provided it receives energy from chlorophyll. Courtesy of Rudi Berera, John Kennis and Rienk van Grondelle.

Rupan_c08.indd 169 8/29/2012 5:36:03 PM


550–600 nm region corresponds to the absorption from S 1 excited states to the range of

higher excited states (S n ) and is called the excited state absorption.

Femtosecond transient absorption spectroscopy requires a complex and expensive

equipment and is not protected from various additional events, such as stimulated emis-

sion, scattering artefacts, and so on. Nevertheless, the method remains an incisive insight-

ful tool to assess the primary light energy conversion events, taking place in the

photosynthetic membrane. This method is therefore unique and promising to aid our

understanding of the molecular energetic design of the light harvesting antenna complex,

the role of single pigments and pigment types in ensuring its remarkable efficiency and

adaptability.

8.3 Spectral and Structural Identity of LHCII Xanthophylls

Whilst chlorophylls possess strong absorption and fluorescence, xanthophylls do not prac-

tically fluoresce. This limits their conventional spectroscopic analysis only to absorption.

However, since there are three different types of xanthophylls attached to LHCII at one

time that differ in absorption and possess three almost equally strong absorption bands,

the identification of these molecules was always a problem. The second derivative absorp-

tion spectrum in the Figure 8.1 (a) reveals at least three xanthophyll bands at 510, 495 and

485 nm. There is also a peak at 457 nm. In order to study the role of these molecules in

light harvesting as well as the significance of their various structures the assignment of

their electronic transitions is of paramount importance. This knowledge provides finger-

print information about each type of molecule and the antenna environment it is associated

with. The essential structural insights obtained for neoxanthin and violaxanthin, described

in Chapter 6, would not be possible to obtain without undertaking the task of development

of the identification methodology. Information about excited states energy levels also

helps to design and interpret kinetic experiments, which probe molecular interactions and

the energetic relationship between xanthophylls and chlorophylls within light harvesting

complexes.

Xanthophylls exhibit strong resonance Raman enhancement. Therefore in the mixture of

spectrally different xanthophylls present in LHCII it seemed to be feasible to achieve a selec-

tive enhancement of the Raman scattering by exciting a single absorption band belonging to

preferentially one xanthophyll species. Moreover, if every xanthophyll of LHCII possesses

a specific resonance Raman spectrum it should be possible to identify the origin of the xan-

thophyll bands in the absorption spectrum (Figure 8.1 ). Indeed the resonance Raman spectra

reveal specific features for all xanthophylls (Figure 8.7 ). Four main regions can be seen in

the xanthophyll Raman spectrum (labelled with a Greek letter ν ). The first and highest fre-

quency region corresponds to C = C stretching vibrations. The second and most complex

region is most influenced by C–C stretching modes coupled to C–H in plane bending/wag-

ging or C–CH 3 stretching vibrations. The third group in the xanthophyll Raman spectrum

reflects CH 3 in-plane rocking vibrations. The fourth and smallest, seemingly featureless

region corresponds to weakly-coupled C–H out-of-plane bending modes. The ν 4 feature

Rupan_c08.indd 170 8/29/2012 5:36:03 PM


would normally be Raman-forbidden for a fully planar configuration of the xanthophyll

molecule. However, as appears later, these modes could become very strong in molecules,

which adopt distorted configuration due to interactions with their environment. Resonance

Raman spectra of all four LHCII xanthophylls reveal differences in the ν 1 frequency, which

normally depends upon the conjugation number. In addition, the neoxanthin transition is

further upshifted reflecting its cis -conformation. The ν 1 region of this xanthophyll possesses

additional bands at 1120, 1132 and 1203 cm −1 characteristic for the 9- cis configuration. The

ν 3 band frequency also differs in these xanthophylls. Finally, ν

4 is small and featureless in all

isolated pigments.

The identification of xanthophylls in LHCII was not a trivial task and required a gradual

approach using samples of progressively increasing complexity. LHCII was first isolated

containing only the internally-bound xanthophylls, neoxanthin and two luteins. The periph-

eral site, V1, was unoccupied. Figure 8.8 (a) displays dependency of the ν 1 Raman band

position upon excitation into various absorption bands in the structure of the absorption

spectrum. Nearly all transitions in the second derivative spectrum were probed. The ν 1

revealed remarkable dependency upon the excitation wavelength bouncing from the ν 1

positions for isolated neoxanthin and lutein that themselves are weakly dependent upon

excitation wavelength. The highest frequency of ν 1 was obtained for excitations near 485

1000

Ram

an In

tens

ity, r

el.

1100 1200

Wavenumber, cm–1

1500 1525 1550

Zea

Vio

Lut

Neo

ν3

ν4

ν2

ν1

cis-peaks

Figure 8.7 Resonance Raman spectra of isolated LHCII xanthophylls, neoxanthin (Neo), Lutein (Lut), violaxanthin (Vio) and zeaxanthin (Zea). Horizontal double arrows indicate the range of variations in n1 and n3 peak positions. Two vertical arrows indicate spectral fingerprints of a cis-conformation. Reproduced from Ruban, A.V. © 2010 Taylor and Francis.

Rupan_c08.indd 171 8/29/2012 5:36:03 PM

920 930 940 950

Wavenumber, cm–1

Monomer

Trimer

Lut 621

450

–0.03

0.00

0.03

0.06

0.09

0.12

0.15

1524

1525

1526

1527

1528

1529

1530

1531

1532

1533

1534

465 480

Resonance wavelength, nm

495 510 525

Ram

an in

tens

ity, r

el.

960 970

Abs

orpt

ion,

rel

.R

aman

ν1,

cm

–1

Neo

Lut

476

466

485

495

510

457

(a)

(b)

Figure 8.8 (a) Dependency of LHCII trimer resonance Raman ν 1 position upon excitation wavelength compared to the structure of the Soret band absorption spectrum. (b) Structure of the LHCII resonance Raman ν 4 region in different oligomeric states. Reprinted from Ruban, A.V. © 2010 Taylor and Francis.

Rupan_c08.indd 172 8/29/2012 5:36:04 PM


and 457 nm bands. Since neoxanthin has the highest ν 1 frequency of all the LHCII xantho-

phylls the measurements led to the conclusion that these maxima belong to 0–0 (zero elec-

tron) and 0–1 (first vibrational) transitions of neoxanthin. Moreover, the near 28 nm spacing

between them is in good agreement with that observed for xanthophylls and carotenoids in

general and measured in vitro and in vivo . Resonance Raman spectroscopy has also revealed

the 9- cis fingerprint features at 1124, 1132 and 1203 cm −1 of neoxanthin in LHCII (see

Figure 6.3). In addition, the ν 3 band position for excitation near 485 nm band is centred at

1006 cm −1 , that is also characteristic for neoxanthin, whilst this band for lutein is positioned

at 1003 cm −1 .

Excitation of the resonance Raman scattering in LHCII trimers near the two longer

wavelength bands at 495 and 510 nm produces resonance Raman spectra having the ν 1

position close to that expected for lutein, that is, ~1527 cm −1 . For the excitation wave-

lengths around 466 and 476 nm bands the ν 1 frequency is also found to be near to that of

lutein. Therefore, it is concluded that 510, 495, 466 and at least part of 476 nm band cor-

respond to the absorption of lutein molecules. Since the wavelength difference between the

first two long-wavelength transitions is only 15 nm it is highly unlikely that they originate

from the same pigment, since the wavelength gap between 0–0 and 0–1 transitions is

almost twice larger (see previous). Therefore, it was suggested that two luteins of LHCII

have different absorption spectra. For the 495 nm absorbing lutein, the suitable 0–1 transi-

tion should correspond to the 466 nm band. For the 510 nm or long-wavelength lutein the

0–1 should be located somewhere on the slope of 476 nm band, most likely at around 482

nm. Since the 510 nm band is almost 50% broader than the 495 nm band the second

derivative spectrum is expected to be of reduced amplitude and poorer resolution. Early

studies using the transient absorption spectroscopy have indicated that the pigment absorb-

ing at 510 nm is closely associated with the short-wavelength chlorophyll a molecules.

Monomerization of the LHCII trimer led to a complete disappearance of this 510 nm band

and parallel enhancement and broadening of the 495 nm transition implying the shift of the

510 nm band down to the 495 nm region. These observations allowed the assignment of the

510 nm transition to lutein 621 (Lutein 2). The β -ring of this xanthophyll is involved in

‘sandwiching’ chlorophyll a 604 with neoxanthin (see Chapter 6). Lutein 621 is also facing

some pigments situated on neighboring monomers in the inner site of the trimer. Figure 8.9

shows the lutein 2 ε -ring is in the van der Waals contact with Chl a 603 of the neighbouring

monomer. All polar oxygen groups of this chlorophyll are positioned closely near the ring.

This electronic perturbation can be a very strong effect in the easily polarizable xantho-

phyll molecules and may be the major cause of the 15 nm (more than 600 cm −1 ) red shift.

This explanation would be consistent with a blue shift of the 510 nm band upon mono-

merization of the trimer.

The 510 nm absorbing species in LHCII reveals a strong negative CD band, implying

that the molecule is in a deformed configuration resulting from an interaction with its

environment. In addition, Stark absorption measurements showed that this species pos-

sesses a very large dipole moment. This is also an indicator of its very specific surround-

ings exerting a polarization effect. Resonance Raman is yet another approach to explore

the configuration of this molecule. Measurements of the resonance Raman spectra excited

Rupan_c08.indd 173 8/29/2012 5:36:04 PM


near 510 nm revealed four clearly defined and pronounced bands in the ν 4 region

(Figure 8.8 b). These are fingerprints of a twisted carotenoid configuration, which can be

completely abolished by monomerization of trimers (Figure 8.8 b). Therefore, this method,

for example, is very useful for assessment of the state of LHCII in vivo at various physi-

ological conditions.

The analysis of the LHCII trimer structure suggests that the interaction of Lut 621 with

Chl a 603 could force the lutein molecule to adopt a twisted configuration (Figure 8.9 ). In

addition, strong interaction with a number of aromatic residues, in particular tryptophan

and phenylalanine, which possess relatively large surface areas, could further promote this

distortion. Lut 621 appears to be more distorted along the carbon backbone than Lut 620

(Lutein 1). The fact that the distortion can be seen at 2.72Å resolution suggests its relatively

large magnitude.

Presence of violaxanthin brings additional complexity to the absorption spectrum of

both thylakoid membranes and isolated LHCII. Differential spectral analysis was applied

for identification of xanthophyll cycle carotenoids, violaxanthin and zeaxanthin.

Figure 8.10 (a) displays the absorption spectra of thylakoid membranes measured before

and after conversion of nearly 80% of violaxanthin into zeaxanthin. The measurement at

liquid helium temperature ensured the highest possible resolution of the spectral structure.

The de-epoxidized- minus -epoxidized difference spectrum is similar to a three maxima/

minima component spectrum of zeaxanthin- minus -violaxanthin in solvents. The absorption

spectrum of zeaxanthin is red-shifted relative to that of violaxanthin. This is due princip-

ally to the presence of 11 conjugated double bonds in zeaxanthin relative to only nine in

violaxanthin. As is evident from the difference spectrum, the 0–0 maxima positions of

zeaxanthin and violaxnthin are localized around 510 and 488 nm, respectively. Therefore,

the resonance Raman signals from these xanthohyplls can be separated by selective excita-

tion near these bands. In order to obtain nearly absolute purity of the spectra of these

xanthophylls it was necessary to calculate difference Raman spectra of membranes (also

Figure 8.9 Intermonomer interaction between lutein 621 (Lut 2) and chlorophyll a603 of the LHCII trimer. Inset shows the view of chlorophyll a603 facing the end group of lutein with all polar oxygen-carrying groups.

Rupan_c08.indd 174 8/29/2012 5:36:04 PM


LHCII) containing violaxanthin or zeaxanthin. These spectra are displayed in Figure 8.10 (b).

The differential Raman method produced spectra similar to those of xanthophyll cycle

carotenoids in pure solvents (compare with Figure 8.7 ). The ν 1 band peaks of violaxanthin

Figure 8.10 (a) Low temperature absorption spectra (Soret region) of thylakoid membranes enriched in violaxanthin (+Vio) and zeaxanthin (+Zea). Dotted line is a zeaxanthin-minus-violaxanthin difference spectrum. Arrows indicate 0–0 electronic transitions of violaxanthin (487 nm) and zeaxanthin (510 nm). (b) Refined resonance Raman spectra of violaxanthin (Vio) and zeaxanthin (Zea) in vivo . Inset shows the zoomed ν 4 region. Reprinted from Ruban, A.V. © 2010 Taylor and Francis.

Wavelength,nm

–0.1

Abs

orpt

ion

(a)

(b)

Wavenumber. cm–1

Ram

an in

tens

ity,r

e.

Rupan_c08.indd 175 8/29/2012 5:36:04 PM


and zeaxanthin spectra are 7 cm −1 apart and in correspondence to the maxima of this band

for isolated zeaxanthin and violaxanthin, respectively. The ν 3 band for zeaxanthin is posi-

tioned at 1003 cm −1 , whilst the one for violaxanthin is up-shifted towards 1006 cm −1 . The ν 4

region in the resonance Raman spectra for violaxanthin and zeaxanthin in vivo reveals a

significant enhancement with the appearance of a number of bands (Figure 8.10 b). The

expanded ν 4 regions in these spectra are shown on the inset in the Figure 8.10 (b). The spec-

trum for zeaxanthin is richer in structure than that for violaxanthin. It contains four bands

and two clearly defined shoulders, and is similar to that of lutein 621 (Figure 8.8 ). The

Raman spectrum of ν 4 for violaxanthin shows only two bands, at 950 and 965 nm, with a

few minor shoulders. The pronounced ν 4 structure is indicative of the xanthophyll distor-

tion in the binding pocket V1. Although the binding site for zeaxanthin in the LHCII has

not been revealed, its strong binding affinity and the complexity of the Raman spectra of

membranes/isolated LHCII shows that this xanthophyll is in a close association with the

complex. Solubilization of photosystem II membranes with detergents and the use of some-

what higher detergent concentrations in the LHCII incubation medium cause a decrease in

the ν 4 amplitude and disappearance of its structural features. Under these conditions, zeax-

anthin becomes largely dissociated from the antenna and it is found to migrate in the free

pigment band on the sucrose gradient. Taken together these data indicate that the in vivo

molecular conformation of the xanthophyll cycle carotenoids relies upon the oligomeric

organization of the antenna. It is interesting to note, the ν 4 region for xanthophyll cycle

carotenoids bound to the minor antenna complexes, CP26 and CP29, reveals little structure

despite the fact that they remain bound in these complexes under higher detergent condi-

tions. Therefore, it is feasible to assume that, the ν 4 fingerprint reflects binding of the xan-

thophyll within a specific site: any dislocation from this site can cause structural relaxation

of the molecule without necessarily inducing its detachment from the protein.

8.4 Plasticity of Light Harvesting Antenna Design: Tailoring the Structure to Optimize the Function

Chapter 7 described a remarkable manifestation of robustness of the LHCII antenna design

using an example of Lhcb2 antisense Arabidopsis plants. In this mutant the normally

monomeric CP26 complex formed trimers and replaced the light harvesting function of

the missing major LHCII antenna complex. This section will describe the findings of some

modifications in pigment composition and states of CP26 trimers that provide interesting

insights into those features of LHCII that ensure its optimized light harvesting function.

The pigment analysis revealed that the homotrimers of CP26 complexes have a Chl a/b

ratio lower than that of the monomeric wild type CP26. This corresponded to the presence

of one extra chlorophyll b molecule in the CP26 trimer. Absorption spectra shown in the

Figure 8.11 (a) clearly reveal a strong enhancement in chlorophyll b band at 650 nm in the

CP26 trimers from the antisense plants in comparison to the monomeric wild type CP26

complex. Calculations based on the increase in the area under the absorption spectrum

also suggest one extra chlorophyll b . Interestingly that the Chl a/b ratio of the CP26/Lhcb3

Rupan_c08.indd 176 8/29/2012 5:36:05 PM


heterotrimer was even lower than that of the CP26 homotrimer and corresponded to the

heterotrimer composition of two CP26 and one LHCII, Lhcb3-based complex. Treatment

with phospholipase was effective in causing monomerization of the CP26 trimer and the

loss of one chlorophyll b molecule. This implied that the extra chlorophyll b binding in the

CP26 is connected to the trimerization process. The cluster of chlorophylls b 601, 606 and

609 involves pigments in adjacent monomers (see Chapter 6). The phospholipid molecule

that has a key role in the stabilization of the trimer is tightly associated with Chl b 601,

which is not H-bonded at either formyl C-7 or carbonyl C-13 as are other chlorophyll b

pigments. It appears that the inclusion of the phospholipid create the binding pocket for

this chlorophyll. It is suggested that trimerization of CP26 recreates some features of this

pocket and causes an extra chlorophyll b to be bound, enabling it to function efficiently in

energy transfer, hence contributing to an extra absorption cross-section.

400 420 440 460 480

Wavelength, nm

500 520 540

0.0

0.5

1.0

1.5

Abs

orpt

ion

2.0620

0.0

0.5

1.0

1.5

2.0

640 660 680 7003-CP26 Monomer

2-CP26 Trimer

1-LHCII

3

2 1

1”

2”3”

510

(a)

(b)

Figure 8.11 Absorption spectra in the red (a) and Soret (b) regions of isolated major (LHCII) and CP26 complex in trimeric and monomeric forms. 1’, 2’ and 3’ are the corresponding second derivative spectra.

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The Soret band in the absorption spectra of LHCII and CP26 trimers appear to be also

very similar and in particular their second derivatives display the presence of the 510 nm

absorption band of the same magnitude (Figure 8.11 b). This band, as was shown in this

chapter arises from lutein 621 (Lutein 2), is a characteristic for the trimeric LHCII state

and is clearly absent from the CP26 monomer, which displayed only the lutein band at

~495 nm. Presence of the red-shifted lutein broadens the light harvesting antenna cross-

section, since it can absorb light quanta with a broader range of energies (colour).

Figure 8.12 illustrates the contribution of the red-shifted lutein to the total amount of

absorbed light in the leaf. Since the absorption is not very strong at >500 nm the sun

light passes through the leaf well, so even the mesophyll cell layers situated closer to the

Figure 8.12 Spectra of the sun light and energy absorbed by the LHCII complex in trimeric and monomeric states. Bottom panel shows the relative leaf penetration depth of the sun light of different spectral regions/colour. (See Plate 8.12 in colour plate section.)

Rupan_c08.indd 178 8/29/2012 5:36:05 PM


back of the leaf can receive it. Appearance of 510 nm absorption band makes sunlight

available for them as well as the upper layer cells that absorb very strongly blue and red

light making it less available for the deeper layers of cells. Hence, 510 nm lutein of

LHCII trimers is important not only for the broadening of the absorption cross-section

but also essential for ensuring light harvesting throughout the whole depth of the leaf.

Calculations suggest that the red-shifted lutein 621 gives approximately 5% of extra

absorption cross-section to the light harvesting antenna. Globally, the extra energy input

given by this pigment into the Biosphere equals to the amount of energy produced by

EU and US altogether.

The Lhcb2 antisense plants lacking the polypeptides of the major LHCII complex

showed not only robust, resilient design of the light harvesting antenna, proving its essen-

tial importance for plant survival but also they demonstrated the antenna plasticity. The

latter is manifested from ability of the antenna protein to adopt extra pigments and mod-

ify the spectral features of existing ones in order to broaden the absorption cross-section,

hence enhancing the light capture capacity of the photosynthetic membrane. Therefore,

the existence of the LHC gene family is an extremely great evolutionary achievement.

The biological function is ensured better when several related genes express similar but

yet distinct proteins that at certain conditions can mimic the functions of each other, a

useful back-up arrangement. The existence of such phenomenon in light harvesting

antenna indicates how important it is in the work of the photosynthetic membrane. In

addition, it is not only the light harvesting function that is ensured by the presence of the

complex gene family, the adaptations to light are also reproduced by the differential

expression of related genes. In the case of Lhcb5 expression employed by the cell in order

to mimic the functions of missing Lhcb1 and 2 proteins, the former is not only trimerized

and incorporated into the PSII supercomplex, it formes the red-shifted lutein (Lutein

621), attaches more chlorophyll b and possesses red-shifted chlorophyll a forms to

enhance the overall cross-section (see the next chapter). In addition, those red-shifted

forms make the novel LHCII antenna spectrally less distinct from PSI antenna, therefore

largely cancelling the spectral requirement of the two photosystems and making state

transitions redundant (see the next chapter). This great response of the LHCII expression

and assembly systems works perfectly, since the Lhcb5-based antenna is not capable of

state transitions at all!

8.5 LHCII Oligomerization: Dynamics of the ‘Programmed Solvent’

8.5.1 Alterations in the Spectral Properties of LHCII

Studies on isolated LHCII complexes mentioned so far have been performed in conditions

of complete solubilization with concentrations of mild detergents enabling them to form

micelles (above critical micelle concentration, cmc ). In this state LHCII trimers are well

separated from each other: the state that is arguably different from that of LHCII in the

studied crystals or, indeed, in vivo , in the protein-crowded photosynthetic membrane (see

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Figure 3.1h). This fact did not concern those who studied isolated LHC complexes using

variety of biophysical techniques for some time. However, eventually the state of LHCII

crossed the attention of Horton’s group (Ruban and Horton, 1992 ), who systematically

studied and documented an interesting phenomenon of the isolated LHCII fluorescence

decrease when trimers formed oligomers (aggregates) upon removal of a detergent. The

latter process was first achieved by dialysis and later by simple dilution or detergent

removal by hydrophobic resins leading to a decrease in the detergent concentration below

cmc levels. Figure 8.13 shows the comparison of fluorescence lifetime (yield) of isolated

diluted chlorophyll solution, trimeric LHCII and LHCII aggregates, obtained by slow

removal of detergent by resin. The chlorophyll fluorescence lifetime/yield of the LHCII

Figure 8.13 Quenching of chlorophyll a fluorescence lifetime in LHCII aggregates. Bottom : negative stain electron microscopy of LHCII aggregates.

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trimer was only 20% lower than that of the diluted chlorophyll, indicating that the

‘programmed solvent’ is very effective in preventing the concentration quenching in

chlorophylls of the complex. When detergent concentration falls below the critical micelle

concentration, LHCII form oligomers/aggregates (Figure 8.13 , bottom). The aggregate size

can vary from tens to hundreds of nanometres. Each particle can contain from several units

to many dozens of LHCII trimers. In the absence of magnesium cations, these aggregates

are two-dimensional, flat particles. Magnesium promotes formation of complex and large

three-dimensional aggregates. However, even the aggregates shown in the Figure 8.13

reveal at least seven times (85%) the quenching of the fluorescence yield. Indeed, rigorous

experiments showed only a loose link between the aggregate size and the fluorescence

decline/quenching. The process was found to be reversible, that is, addition of detergent

caused gradual restoration of the fluorescence lifetime/yield eventually to that of completely

solubilized trimers. It was also shown that the aggregation process did not practically affect

the intactness of the trimeric complex or its properties, confirming strongly reversible

character of the aggregation process. The latter was found to be promoted by a variety of

factors that included magnesium cations, low pH and hygroscopic media such as glycerol.

LHCII aggregation was found to have profound effects on the properties of bound

pigments. First of all, the fluorescence spectrum of the aggregated complex taken at the

liquid nitrogen temperature (77K) revealed a striking feature: a broad long-wavelength

fluorescence band at 700 nm (F700, Figure 8.14 ). This band was used as a fingerprint of

aggregation and found to be reasonably well-correlated with the extent of the fluorescence

quenching. The long wavelength fluorescence was largely absent in the room temperature

fluorescence spectrum of aggregates, indicating it possessed a strong temperature

dependence. Thorough investigation of the latter provided an insight into the structure of

F700 band. It revealed heterogeneity, existence of at least five different emission bands

from 685–700 nm each differently depending upon temperature. It was initially suggested

660 680 700 720

Wavelength, nm

740 760 780

Trimer

Flu

ores

cenc

e, r

el.

F680 F700

Aggregate

LHCII

Figure 8.14 Comparison of the low-temperature (77 K) fluorescence spectra of LHCII trimers and aggregates.

Rupan_c08.indd 181 8/29/2012 5:36:07 PM


that these bands reflect the heterogeneous nature of LHCII aggregates and are likely to

belong to various minor fluorescing chlorophyll associates that are formed in the interface

of interacting trimers within the aggregate. One of the reasons for such a suggestion was

the absence of absorption bands that corresponded to the red-shifted fluorescence. Indeed,

the absorption spectrum of aggregates revealed a little change, partially due to the selective

light scattering artefacts that occur on the relatively large particles of aggregates. In

addition, a small red shift of the major red-absorbing chlorophyll a (terminal emitter) and

small alterations in chlorophyll b and xanthophyll absorption was observed upon

aggregation. Figure 8.15 shows the aggregated- minus -trimer difference spectrum of LHCII

displaying alterations in the mentioned pigments with the largest being the long-wavelength

chlorophyll a (appearance of 681 nm band). Polarized light spectroscopy indicated the

possibility of altering the coupling between some chlorophyll b molecules (645–655 nm

region), slight reorientation of the terminal emitter chlorophylls and xanthopylls (more

parallel to the membrane plane) and change in the conformation of a subpopulation of long

wavelength chlorophyll a (positive circular dichroism band at ~680 nm and negative band

at 438 nm). All mentioned changes observed indicated alterations in chlorophyll

environment of the LHCII trimer, specifically in the terminal emitter locus, Chl a 610–612.

Analysing these early measurements Ruban and coworkers (Ruban et al ., 1992 ) proposed

the formation of chlorophyll-chlorophyll associates that possess very low fluorescence

yield and therefore quench fluorescence of the trimer. Indeed, interpigment associates

often found to possess red-shifted spectra and low fluorescence. The scenario was similar

to that of the concentration quenching discussed in Chapter 5. As if the ‘programmed

solvent’ of LHCII sized its function to dissolve, separate pigments and prevent their

interactions that can lead to the excitation/fluorescence quenching. The latter is obviously

an unwanted effect because it does undermine the antenna function. Shortening of the

excited state lifetime due to nonradiative dissipation will certainly compete with the energy

transfer channel that is essential for light harvesting in the photosystem unit. The ideas of

Ruban have been recently taken further by Holzwarth ( 1995 ) who proposed that the

Figure 8.15 Aggregation-induced changes in the 77 K absorption spectrum of LHCII.

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chlorophyll-chlorophyll associates in LHCII aggregates have a charge transfer character.

The latter is characterized by the enhanced coupling between excited and ground states and

therefore can explain the reduced excited state lifetime as well as the red shift and strong

temperature dependence of the fluorescence yield (see previous). Recent experiments by

van Grondelle’s group (Wahadoszamen et al ., 2012 ) confirmed this idea as well as finding

a chlorophyll-xanthophyll charge transfer state in the quenched LHCII and the minor LHC

complexes . These chlorophyll-xanthophyll interactions have been also proposed as a cause

of the enhancement in energy dissipation. The idea comes from the fact that promotion of

chlorophyll-xanthophyll interactions can potentially quench chlorophyll fluorescence

simply because the xanthophyll S 1 excited state lives only about 10 ps, a perfect nonradietive

energy quencher. The only one feature that complicates this possibility is that S 1 is a

symmetry-forbidden state. Hence, it does not reveal any absorption because the transition

dipole moment is zero and therefore the classic energy transfer from the interacting

chlorophyll to this state, at least in the dipole approximation, should not be possible.

Nevertheless, van Grondelle and coworkers (Ruban et al ., 2007 ), using the transient

absorption spectroscopy (see Figure 8.6 ) revealed the opening an energy transfer channel

from chlorophyll a of the terminal emitter (a610-612) to lutein 620 (Lut 1) caused by

LHCII aggregation. It was noted that the energy transfer rate from chlorophyll to lutein was

much slower than the excited state lifetime of the xanthophyll.

8.5.2 Structural Changes within LHCII

The design of LHCII shows a remarkable flexibility manifesting in the existence of a

switch-like property that can alter the efficiency of antenna function. It appears as if the

changes in the LHCII structure upon aggregation promote the formation of multiple energy

dissipation pathways that involve chlorophylls as well as xanthophylls. It is not surprising,

therefore, that LHCII aggregates have become a new model for various insightful studies

during the last 20 years. These studies aimed to gain more information about the nature of

such flexible behaviour of the complex and the physics of the enhanced excited state non-

radiative dissipation (quenching). Some specific/microscopic information about the pig-

ment structural states in the aggregated LHCII was obtained using the resonance Raman

spectroscopy. The method revealed a few striking features, that emerged upon aggregation.

One of them was the alteration in the hydrogen bonding patterns of a specific fraction of

chlorophyll a and b . New hydrogen bonds were fund to form in LHCII aggregates, most

explicitly to the formyl group of chlorophyll b (Figure 8.16 a). Another striking feature that

was revealed was a strong twist-like configurational alteration in neoxanthin and lutein 620

(Lut 1) xanthophylls (Figure 8.16 b). Establishment of hydrogen bond(s) and twisting of

xanthophylls structure reflect some small structural changes within the LHCII protein. The

thermodynamic studies found that they correspond to the enthalpy change of ~85 kJ mol −1

(several hydrogen bonds) and more than 30 times higher than that of kT at the room tem-

perature. However, these energy changes are nearly five times smaller than those that occur

during the unfolding of the complex and they do not manifest in the inhibition of energy

transfer between chlorophyll b and a (compare Figures 8.3 and 8.14 ). Therefore, it was

proposed that the quenching of fluorescence in LHCII corresponded to the new state of the

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16000

200

Ram

an s

catte

ring,

rel

.

400

600

(a)

(b)

1610 1620 1630

Wavenumber, cm–1

LHCII chlorophyII

1640 1650 1660

Trimer

952

Aggregate

Aggregate

Trimer

1640

LHCII xanthophyll

940

0.0

0.2

0.4

0.6

Ram

an in

tens

ity, r

el. 0.8

1.0

1.2

950 960

Wavenumber, cm–1

970 980

Figure 8.16 Resonance Raman pigment ‘fingerprints’ associated with aggregation of LHCII. (a) Chlorophyll carbonyl region. (b) Xanthophyll n 4 region. Arrows indicate appearance of the new spectral features associated with fluorescence quenching.

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complex different from the denaturation and having specific alterations in the pigments

associated with the promotion of their interactions with each other. This situation suggests

that the protein in aggregated state is no longer capable of keeping the pigments apart from

each other (solving) and preventing the establishment of the type of interactions that cause

excited state/fluorescence quenching.

Observation of structural alterations within LHCII pigments prompted a discussion about

their origins as well as the role of the protein aggregation per se in the observed changes. At

first it was believed that the aggregation is absolutely essential for the fluorescence quench-

ing and the changes observed occur on the interface between interacting trimers within the

LHCII aggregate. However, a few lines of experimental evidence emerged recently that

pointed out that the protein aggregation is not the primary cause but rather a consequence of

the trimer/monomer transition into the structural quenching state. Firstly, a high fluorescence

quenching was possible to obtain using high hydrostatic pressure (5–6 kBar) at the detergent

concentration well above the cmc and the absence of LHCII aggregation. The quenching was

reversible and followed by the changes in absorption spectra similar to those shown in the

Figure 8.15 . Secondly, the rate of transition of LHCII into the quenching state was found to

be reciprocally related to the protein concentration, which is opposite to what was expected

if the aggregation was the direct cause of the quenching. Thirdly, the quenching was obtained

in LHCII trimers incorporated into the PAA or gelatine gels to prevent aggregation

(Figure 8.17 a). Gels were incubated in the low detergent buffer to induce quenching. Return

to the high detergent buffer reversed the effect. Whilst it was impossible to move the polym-

erized aggregates of LHCII along the gel by the electric field, in-gel quenched LHCII moved

as quickly as solubilized unquenched trimers (Figure 8.17 b). This experiment suggested that

the quenching of LHCII incorporated into PAAG was not associated with the protein aggre-

gation. This quenching was followed by fluorescence, absorption and circular dichroism

spectral changes resembling those which accompanied LHCII aggregation.

Fourthly, the recovery of LHCII fluorescence quenching induced in gels was strongly

sensitive to the addition of cross-linkers, like glutaraldehyde (Figure 8.17 a). This cross-linker

is able to covalently bind to the two sites of a protein that are closely associated to each other

and strongly restrict its intrinsic movement/conformational dynamics. The cross-linker,

therefore, builds an entire ‘web’ within the protein ‘freezing’ greatly its conformation.

Hence, once again, the fluorescence behaviour of LHCII links to the structural state of the

complex. Finally, the most recent evidence comes from the work that uses His-tag attached

to LHCII. When the tagged complex is immobilized at very low concentration on the

Ni-containing column to completely prevent possibility of aggregation and exposed to the

low-detergent buffer it reveals very strong, reversible fluorescence quenching. All the afore-

mentioned experiments indicate that the intrinsic dynamics of LHCII trimer or indeed mono-

mer is responsible for the switching of it between the dissipative and highly efficient light

harvesting states. The thermodynamic nature of the structural switch within the LHCII seems

to be almost entirely entropically-driven. Indeed, whilst the enthalpy of the transition into the

dissipative state was found to be around 85 kJ mol −1 (see previous), the free energy difference

between the efficient and dissipative states estimated from the high pressure experiments was

only about 7 kJ mol −1 . Whilst the two LHCII states are almost isoenergetic at the given

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conditions, the switching between them encounters an energy barrier the crossing of which

heavily relies upon the entropic factor.

One of the obvious experiments relating the described phenomenon of fluorescence

quenching in LHCII to its structure was to study crystals that were used to obtain the

atomic model. These crystals were studied using the measurements of fluorescence life-

time (see previously) combined with confocal microscopy. The method, called FLIM,

fluorescence lifetime imaging, was first employed by Ruban and van Amerongen (Pascal

et al ., 2005 ) to study LHCII crystals. The measurements produced images of LHCII crys-

tals that reflected the spatial redistribution of chlorophyll fluorescence lifetime that was

found to be highly homogenous, reflecting the order of LHCII complexes in the crystal and

the absence of damage to the structure. Figure 8.18 shows FLIM image of the typical

Figure 8.17 (a) Induction of fluorescence quenching in LHCII incorporated into PAA gel. (b) Gel electrophoresis run of the in-gel-quenched LHCII showing its high mobility and likely trimeric state.

(a)

Aggregated

Quenchedin gel

Solubilisedunquenched

LHCIIBefore run

Trimers

Aggregates

Run direction

After run(b)

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0.8 ns 1.1 ns

864Time, ns

Flu

ores

cenc

e, r

el

200

1000

2000

LHCII trimer

LHCII crystal

Figure 8.18 Quenching of chlorophyll a fluorescence lifetime in a single LHCII crystal using FLIM technique. The image is shown in false colours defining the fluorescence lifetime as presented by the colour lifetime scale below the image. Bottom: average fluorescence decay profiles for trimers and crystals of LHCII. Reprinted by permission from Macmillan Ltd Publishers, Pascal et al . © 2005 Nature Publishing Group. (See Plate 8.18 in colour plate section.)

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hexagonal crystal used for the structural studies. The average fluorescence lifetime traces

are shown below it. It was remarkable to find out that the fluorescence of LHCII in

crystalline state was significantly quenched with the average lifetime of about 0.86 ns in

comparison to 4 ns of the isolated trimers (Figure 8.13 ). This corresponds to about 78% of

quenching, slightly less than the quenching observed in some LHCII aggregates. The reso-

nance Raman features that accompany quenching in LHCII described previously

(Figure 8.16 ) were all present in the crystals, suggesting that the nature of quenching in

them is similar to that of LHCII aggregates.

A novel technique of the fluorescence measurements was recently employed to

further study the properties of the structural switch within LHCII. This is the single

molecule fluorescence spectroscopy. The method uses confocal microscope to detect

and visualize fluorescence from single molecules. Although the special resolution of the

optical microscope is limited to a few hundreds of nanometres it is possible to see the

broad ‘aura’ of the single LHCII trimer fluorescence depicted as a spot of a fraction of

a micron. Figure 8.19 (a) shows a typical image of the surface where LHCII trimers were

immobilized and illuminated to induce chlorophyll a fluorescence which is registered in

spots originating from single complexes. It was found that even in the presence of high

detergent concentration the fluorescence level of a single trimer reveals occasional

spontaneous rapid decrease and returns back to normal (Figure 8.19 b). This ‘blinking’

of the emission is considered by van Grondelle and coworkers (Kruger et al ., 2011) to

be a reflection of the inherent intrinsic disorder within the LHCII protein. On the one

hand, the complex is very densely compacted with pigments; on the other hand, it is

constantly in a rather dynamic mode responding to the entropy input within and from

the environment. The structural organization of the complex (order) limits the number

of possible accessed conformational states, some of which possess low fluorescence.

Overall, throughout billions of complexes in the macroscopic experiment this blinking

behaviour will be averaged into a certain fluorescence level/lifetime. Interestingly, van

Grondelle and coworkers found that the blinking frequency as well as the fluorescence

intensity of unquenched and quenched states was controlled by a number of factors such

as detergent concentration, pH and, most importantly, the presence of xanthophyll cycle

carotenoids, violaxanthin and zeaxanthin. Low detergent concentration and low pH

enhanced the frequency of occurrence of the low fluorescence states suggesting the

presence of the quenching switch control within a single LHCII complex. It is likely that

these factors affect the entropically-driven energy barrier between unquenched and

quenched fluorescence states. The mentioned factors can, for example, inflict certain

minor structural alterations within the protein that will make more probable or

improbable accessing of certain conformations/fluorescence levels. Hence, the nature

invented a largely self-reliant molecular mechanics that is based upon a several

fundamental principles which include microscopic dimensions, highly cooperative and

semi-rigid entropically-driven dynamic structure, properties of which can be modulated

by cofactors and environmental effects. Once again, a biomolecule displayed a

compromise between order and disorder as well as the great extent of autonomy and

responsiveness in its dynamic behaviour. The significance of the latter will become

apparent in Chapter 9.

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8.6 Kinetics of the Collective LHCII Transition into the Dissipative State: Exploring ‘The Switch’ Control

The properties of the transition of LHCII into the dissipative, inefficient state were studied

in a great detail in solution. It was observed that acidification was one of the major driving

factors of this transition. However, at detergent concentration above cmc the fluorescence

06 5 4 3 2

1 0 01

23

Coordinate (μm)Coordinate (μm)

45 6

20

40

60

80F

L (C

ount

s/3

ms)

0

0 5 10 15 20 25

Illumination time (s)

30 35 40 45

20

40

Flu

ores

cenc

e in

tens

ity (

c/10

ms)

60

80

(b)

(a)

Figure 8.19 Single molecule fluorescence spectroscopy of LHCII trimers. (a) Imaging and quantifying fluorescence of single particles. (b) Observing sharp fluctuations in fluorescence intensity within the single LHCII trimer. Reprinted with permission from Kruger et al . © 2011 American Chemical Society.

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quenching was very small (Figure 8.20 ). Incubation of LHCII in the buffer with detergent

concentration below cmc revealed relatively slow spontaneous fluorescence quenching as

well as made the complex more responsive to acidification that prompted fast transition

into the significantly quenched state (compare traces (1) and (2) in Figure 8.20 ). The iso-

lated LHCII used in the experiments did not contain any violaxanthin or zeaxanthin

attached to the peripheral V1 binding site. The isolation method, isoelectric focussing,

stripped nearly all extrinsic xanthophylls, as their binding affinity was found much lower

than that of the intrinsic xanthophylls, lutein and neoxanthin (see Chapter 6). Experiments

3 and 4 shown in the Figure 8.20 exploited the fact of the empty V1 site and weak binding

of xanthophyll cycle carotenoids there in order to see if their rebinding could affect the

quenching behaviour of LHCII. The results for violaxanthin and zeaxanthin effects were

remarkably contrasting. Whilst zeaxanthin addition slightly accelerated the spontaneous

and pH-induced quenching, violaxanthin significantly inhibited the kinetics and ampli-

tude of both processes. It was noted that also zeaxanthin had an effect on the quenching

kinetics it did not enhance its amplitude. The maximum extent of quenching of 4–5 times

within 1 min after acidification with pH = 5.7 was found to be the same here as in the con-

trol. The discovered ability of the peripherally-bound xanthophyll cycle carotenoids to

modulate ‘the switch’ that changes the LHCII efficiency was a remarkable observation

made by Horton’s group that provided first insights into the physiological relevance of the

quenching behaviour of isolated antenna complexes and the role of xanthophyll cycle in

the photosynthetic membrane.

1 min

+DM Control +Vio +Zea

Low detergentH+

1 2 3 4

Figure 8.20 Induction of quenching in isolated LHCII by acidification (pH = 5.5) at detergent ( b -DM) concentration above (1) and below (2-4) cmc. LHCII was isolated with an empty peripheral V1 xanthophyll binding site. For (3) and (4) violaxanthin and zeaxanthin were added before acidification.

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The pH requirement for the observed quenching in LHCII was studied by means of titra-

tions: a broad range of pH was used to induce the quenching, which was then plotted as a

function of pH. Figure 8.21 displays pH titrations for LHCII incubated with violaxanthin

or zeaxanthin. This work showed that the xanthophyll cycle carotenoids mostly effect the

pH requirement for ‘the switch’. Presence of zeaxanthin makes LHCII more sensitive to

acidification with pK, the protein-proton association constant, of about 6.5. Presence of

violaxanthin requires more acidic pH to induce the same amount of quenching as with

zeaxanthin. The pK value of quenching with violaxanthin was about 5.0. These titrations

revealed yet another remarkable property of the LHCII complex, the existence of a mecha-

nism that alters its dynamic behaviour relative to pH of the medium. Affinity to protons was

discovered to be a changeable property of the antenna complex.

Further exploration of ‘the switch’ emerged from the studies of the quenching kinetics

induced at different pH. Not only did the low pH enhance the quenching, it accelerated the

transition into the quenched state. Figure 8.22 (a) displays two quenching kinetics induced

at pH 8.0 (control, spontaneous quenching) and a low pH (5.7). The theoretical fitting of

the experimental data established that the quenching process obeys not exponential but

rather hyperbolic decay kinetics. Reciprocal transformation of the kinetic data confirmed

this discovery, since it produced nearly perfect fits to the linear function of time

(Figure 8.22 b). The gradient of the linearized kinetic functions presented in Figure 8.22 (b)

correspond to the rate constants of the switching process. The hyperbolic quenching kinet-

ics in LHCII (as well as the minor PSII antenna complexes) suggested that the structural

‘switch’ into the dissipative state obeys the binary type of reaction: interaction of the two

40.0

0.1

Flu

ores

cenc

e qu

ench

ing

0.2

0.3

0.4

0.5

0.6

0.7

0.8

5 6

pH

7 8

+Vio

+Zea

Figure 8.21 pH titrations of fluorescence quenching in isolated LHCII with added zeaxanthin (+Zea) or violaxanthin (+Vio). The quenching is a dimensionless parameter, defined as (Fmax-Fq)/Fmax, where Fmax is the initial unquenched level of fluorescence, Fq is the fluorescence level after quenching.

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identical fluorescence domains/pigments are required to display the hyperbolic kinetics of

the transition into the quenched state. The simultaneous disappearance/quenching of the

two fluorescing or contributing to the fluorescence (transferring energy to the terminal

emitter) domains of pigments was required to explain the observed character of kinetic

behaviour. At present, the identity of putative domains remains unknown.

An attempt was made by Ruban and coworkers to explain the effect of violaxanthin and

zeaxanthin upon the fluorescence quenching in isolated LHCII (Ruban and Johnson,

2010 ). Xanthophyll hydrophobicity as well as the rigidity and orientation of the head

groups have been proposed to be key structural features affecting the behaviour of the

structural ‘switch’ within the complex. These properties could be empirically quantified

using the solubility of the isolated xanthophylls in water/ethanol mixtures. An increase in

the ethanol content decreases polarity and increases solubility of hydrophobic molecules.

00.00

0.05

0.101/F

(t)–

Fu)

Flu

ores

cenc

e, r

el.

0.15

0.20

0.25

0

10

20

30

40

50

60

70

80

90

100

30 60Time /s

90

5.7F(t)

F(t)8.0

8.05.7

(a)

(b)

Figure 8.22 Analysis of LHCII fluorescence quenching kinetics at pH 8.0 (spontaneous) and 5.7. (a) Original data. (b) Linearization of quenching kinetics data and fits with hyperbolic decay function with reciprocal transformation, where Fu is the level of unquenched fluorescence obtained from the hyperbolic fit (for more details see the text).

Rupan_c08.indd 192 8/29/2012 5:36:14 PM


The solubility titrations for all LHCII xanthophylls are presented in Figure 8.23 . When a

xanthophyll becomes insoluble it aggregates and drastically changes its absorption spec-

trum. Hence, the solubility can be relatively easily quantified in this experiment. It was

found that the most hydrophilic of all LHCII xanthophylls are neoxanthin, then violaxan-

thin. Zeaxanthin was found to be the most hydrophobic of all the xanthophylls. Therefore,

de-epoxidation of violaxanthin was proposed to have a drastic effect on the LHCII

V1-associated xanthophyll hydrophobicity, binding affinity and the binding locus itself.

This structural alteration was proposed to have an influence on the structural ‘switch’ that

controls the efficiency of LHCII antenna. Since the LHCII structure with bound zeaxan-

thin is unknown it is not possible to accurately analyse the mechanistic effect of zeaxanthin

upon the LHCII structure and dynamics.

The role of intrinsic xanthophylls in the dynamic behaviour of the LHCII ‘switch’ was

also explored using the complexes isolated from different xanthophyll biosynthesis

Arabidopsis mutants using the fluorescence lifetime spectroscopy, TCSPC (for the method

details see previous). LHCII complexes containing neoxanthin and violaxanthin only

(NVVV and NVVZ compositions: neoxanthin, two violaxanthins replacing two luteins at

L1 and L2 sites and extrinsically-bound violaxanthin or zeaxanthin) were isolated and

studied in addition to those containing the wild type levels of xanthophylls. Figure 8.24

displays the results of this study. At high detergent concentration the studied complexes

revealed differences in the fluorescence lifetime yield depending on the xanthophyll com-

position. Those that contained more hydrophobic xanthophylls possessed shorter fluores-

cence lifetime, that is, were more quenched, indicating that both peripheral and intrinsic

xanthophylls affect the LHCII structure and hence the chlorophyll excited state lifetime.

At low detergent concentration and low pH, LHCII was ‘switched’ into the quenched

state (aggregated). Again, the extent of quenching depends upon the xanthophyll compo-

sition. More hydrophilic/polar xanthophylls partially inhibited ‘the switch’ whilst more

200

1

2

Dis

solv

ed /A

ggre

gate

d, r

el.

3

4

30 40 50

Ethanol, %

60 70 80

Zea

LutVio

Neo

Figure 8.23 Quantifying differential solubility of LHCII xanthophylls in water/ethanol mixtures. Arrows indicate the percentage of ethanol corresponding to the point of equilibrium between aggregated and dissolved xanthophyll.

Rupan_c08.indd 193 8/29/2012 5:36:15 PM


hydrophobic promoted it. Indeed, the fluorescence lifetime was the shortest, when the

complexes contained lutein and zeaxanthin instead of violaxanthin. The X-axis of the plot

shown in Figure 8.24 was constructed using the xanthophyll hydrophobicity parameter

(H-parameter). This was calculated taking the percentages of ethanol at equilibrium point

between aggregated and dissolved (see Figure 8.23 ) for all xanthophylls of each complex

and averaging them. For example, for the LHCII complex that possessed NVVV xantho-

phyll composition, the H-parameter was calculated as (32 + 44 + 44 + 44)/4 = 41. For

LHCII with NLLZ composition the H-parameter will be (32 + 50 + 50 + 64)/4 = 49. The

generic effect of xanthophylls regardless of their binding site on the LHCII ‘switch’ sug-

gests that the structural change involved could relate to all xanthophyll binding domains.

This is not surprising since the complex is very tightly assembled with helixes and pig-

ments and small alterations/fluctuations in its structure are likely to be reflected upon the

entire assembly.

References

Holzwarth , A.R. ( 1995 ) Time-resolved fluorescence spectroscopy . Methods Enzymol. , 246 , 334 – 362 . Kruger , T.P.J. , Ilioaia , C. and van Grondelle , R. ( 2011 ) Fluorescence Intermittency from the Main

Plant Light-Harvesting Complex: Resolving Shifts between Intensity Levels . J Phys. Chem. B , 115 , 5071 – 5082 .

Figure 8.24 Dependency of LHCII fluorescence lifetime upon xanthophyll composition at high (top) and low detergent concentration and pH = 5.7 (bottom). The x -axis represents the xanthophyll hydrophobicity parameter (for details of calculation see the text).

Rupan_c08.indd 194 8/29/2012 5:36:16 PM


Pascal , A.A. , Liu , Z. , Broess , K. , et al . ( 2005 ) Molecular basis of photoprotection and control of photosynthetic light-harvesting . Nature , 436 , 134 – 137 .

Ruban , A.V. ( 2009 ) Identification of carotenoids in photosynthetic proteins: xanthophylls of the light harvesting antenna , in: Carotenoids: Physical, Chemical and Biological Functions and Properties . J.T . Landrum (ed.), CRC Press, Florida International University , pp. 113 – 136 . Taylor & Francis .

Ruban , A.V. , Berera , R. , Ilioaia , C ., et al . ( 2007 ) Identification of a mechanism of photoprotective energy dissipation in higher plants . Nature , 450 , 575 – 578 .

Ruban , A.V. and Johnson , M.P. ( 2010 ) Xanthophylls as modulators of membrane protein function . Arch. Biochem. Biophys. , 504 , 78 – 85 .

van Amerongen , H. and van Grondelle , R. ( 1995 ) Transient absorption spectroscopy in study of pro-cesses and dynamics in biology . Methods in Enzymology , 246 , 201 – 226 .

Wahadoszamen , M. , Berera , R. , Ara , A.M. , et al . ( 2012 ) Identification of two emnitting sites in the dissipative state of the major light harvesting antenna . Phys. Chem. Chem. Phys. , 14 , 759 – 766 .

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Blankenship , R. ( 2002 ) Molecular Mechanisms of Photosynthesis . London : Blackwell Science . Clayton , R.K. ( 1970 ) Light and Living Matter, Volume 1: The Physical Part . New York : McGraw-Hill . Clayton , R.K. ( 1970 ) Light and Living Matter, Volume 2: The Biological . New York : McGraw-Hill . Garab , G. and van Amerongen , H. ( 2009 ) Linear dichroism and circular dichroism in photosynthesis

research . Photosynth. Res. , 101 , 135 – 146 . Hall , D.O. and Rao , K.K. ( 1995 ) Photosynthesis . Cambridge : Cambridge University Press . Hipkins , M.F. and Baker , N.R. (eds) ( 1986 ) Photosynthesis: Energy Transduction: A Practical

Approach. Practical Approach Series . Oxford : IRL Press Limited . Ilioaia , C. , Johnson , M. , Horton , P. and Ruban , A.V. ( 2008 ) Induction of efficient energy dissipation

in the isolated light harvesting complex of photosystem II in the absence of protein aggregation . J. Biol. Chem. , 283 , 29505 – 29512 .

Kruger , T.P.J. , Ilioaia , C. and van Grondelle , R. ( 2011 ) Fluorescence Intermittency from the Main Plant Light-Harvesting Complex: Resolving Shifts between Intensity Levels . J. Phys. Chem. B , 115 , 5071 – 5082 .

Lakowicz , J.R. ( 2006 ) Principles of Fluorescence Spectroscopy . New York : Springer . O’Connor , D.V.O. ( 1984 ) Time-Correlated Single Photon Counting . London : Academic Press . Landrum , J. (ed.) ( 2009 ) Carotenoids: Physical, Chemical and Biological Functions and Properties .

Florida International University : CRC Press . Novoderezhkin , V.I. , Palacios , M.A. , van Amerongen , H. and van Grondelle , R . ( 2005 ) Excitation

dynamics in the LHCII complex of higher plants: modeling based on the 2.72 Å crystal struc-ture . J. Phys. Chem ., 109 , 10493 – 10504 .

Ruban , A.V. , Calkoen , F. , Kwa , S.L.S. , et al . ( 1997 ) Characterization of the aggregated state of the light harvesting complex of photosystem II by linear and circular dichroism spectroscopy . Biochim. Biophys. Acta , 1321 , 61 – 70 .

Ruban , A.V. , Duffy , C.D.P. and Johnson , M.P. ( 2012 ) Natural light harvesting: principles and envi-ronmental trends . Energy and Environmental Science , 4 , 1643 – 1650 .

Ruban , A.V. , van Grondelle , R. , Horton , P. and Dekker , J.P. ( 1995 ) Temperature dependence of chlo-rophyll fluorescence from the light harvesting complex II of higher plants . Photochem. Photobiol. , 61 , 216 – 221 .

Ruban , A.V. , Horton , P. and Young , A.J. ( 1993 ) Aggregation of higher plant xanthophylls: differences in absorption spectra and in the dependency on solvent polarity . J. Photochem. Photobiol. , 21 , 229 – 234 .

Ruban , A.V. and Horton P. ( 1992 ) Mechanism of Δ pH-dependent dissipation of absorbed excitation energy by photosynthetic membranes. I. Spectroscopic analysis of isolated light harvesting complexes . Biochim. Biophys. Acta , 1102 , 30 – 38 .

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Ruban, A.V. and Horton , P. ( 1999 ) The xanthophyll cycle modulates the kinetics of nonphotochemical energy dissipation in isolated light harvesting complexes, intact chloroplasts and leaves . Plant Physiol. , 119 , 531 – 542 .

Ruban, A.V. , Lee , P.J. , Wentworth , M. , et al . ( 1999 ) Determination of the stoichiometry and strength of binding of different xanthophylls to the photosystem II light harvesting complexes . J. Biol. Chem. , 274 , 10458 – 10465 .

Ruban, A.V. , Pascal , A. and Robert , B. ( 2000 ) Xanthophylls of the major photosynthetic light- harvesting complex of plants: identification, conformation and dynamics . FEBS Lett. , 477 , 181 – 185 .

Ruban , A.V. , Pascal , A.A. , Lee , P.J. , et al . ( 2002 ) Molecular Configuration of Xanthophyll Cycle Carotenoids in Photosystem II Antenna Complexes . J. Biol. Chem. , 277 , 42937 – 42942 .

Ruban, A.V. , Robert , B. and Horton , P. ( 1995 ) Resonance Raman spectroscopy of photosystem II light-harvesting complex of green plants. A comparison of trimeric and aggregated states . Biochemistry , 34 , 2333 – 2337 .

Ruban , A.V. , Solovieva , S. , Lee , P.J. , et al . ( 2006 ) Plasticity in the composition of the light harvesting antenna of higher plants preserves structural integrity and biological function . J. Biol. Chem. , 281 , 14981 – 14990 .

Ruban, A.V. , Young , A. and Horton , P. ( 1994 ) Modulation of chlorophyll fluorescence quenching in isolated light harvesting complex of photosystem II . Biochim. Biophys. Acta , 1196 , 123 – 127 .

Ruban, A.V. , Young , A. , Pascal , A. and Horton , P. ( 1994 ) The effects of illumination on the xantho-phyll composition of the photosystem II light harvesting complexes of spinach thylakoid mem-branes . Plant Physiology , 104 , 227 – 234 .

Ruban, A.V. , Young , A.J. and Horton , P. ( 1996 ) Dynamic properties of the minor chlorophyll a / b binding proteins of photosystem II - an in vitro model for photoprotective energy dissipation in the photosynthetic membrane of green plants . Biochemistry , 35 , 674 – 678 .

Santabarbara , S. , Horton , P. and Ruban , A.V. ( 2009 ) Comparison of the thermodynamic landscapes of unfolding and formation of the energy dissipative state in the isolated light harvesting com-plex II . Biophys. J. , 97 , 1188 – 1197 .

van Oort , B. , van Hoek , A. , Ruban , A.V. and van Amerongen , H. ( 2007 ) The equilibrium between quenched and non-quenched conformations of the major plant light-harvesting complex studied with high-pressure time-resolved fluorescence . J. Phys. Chem. B , 111 , 7631 – 7637 .

Valkunas , L. , Trinkunas , G. , Chmeliov , J. and Ruban , A.V. ( 2009 ) Modeling of exciton quenching in photosystem II . Phys. Chem. Chem. Phys. , 11 , 7576 – 7584 .

Wentworth , M. , Ruban , A.V. and Horton , P. ( 2001 ) Kinetic analysis of non-photochemical quench-ing of chlorophyll fluorescence. II. Isolated light harvesting complexes . Biochemistry , 40 , 9902 – 9908 .

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‘To know that we know what we know, and that we do not know what we do not know, that is true knowledge ...’

Confucius

The almost-explosive (on the evolutionary scale) emergence of oxygenic photosynthesis

and its success and impact on our Biosphere is a phenomenal event that relies first of all

upon the efficient design and adaptability to the changing environmental conditions of the

molecular machinery of the photosynthetic membrane. The intensity and energy of light

radiation that defines its colour (hv) reveals very large temporal and spatial variations. For

example, the light intensity exposure of plants grown in the deep shade of the tropical forest

corresponds to the daily available photon flux 100 times lower than that available for plants

exposed to direct sunlight. Plants grown in shade environments receive light of a very

different spectral quality/colour from that received by plants grown on the full sunlight. This

is due to the process of filtering of light by canopy, so the light that passes through it becomes

enriched in the far red of the spectrum of sunlight. Plants are also often exposed to rapid and

irregular changes in light intensity. These changes could be caused by clouds shielding the

sun or sudden shading by other plants in crowded communities that often take place in

natural environments. In addition, diurnal changes in light quality and quantity regularly

modulate the light input into the photosynthetic membrane of plants on a day- to-day basis.

It is not surprising, therefore that the evolution selected various multilevel and mechanisti-

cally diverse types of adaptations of the photosynthetic organisms to the only source of

energy, light: a central factor supporting life. Optimization of light input became the central

adaptation that drove remarkable biodiversity of nature and largely determined the evolution

of all life forms on our planet.

Adaptations of the Photosynthetic Membrane to Light

9

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9.1 The Need for Light Adaptations and their Various Strategies

The efficient harnessing of light energy, even in a very shaded environment, is ensured

by the existence of the light harvesting antenna (see Chapter 5). However, the drastic

fluctuations of light mentioned above create the requirement for mechanisms that

ensure ‘smooth’ inputs of energy into the photosynthetic apparatus for its optimum and

safe utilization. A number of limiting factors, restricting the achievement of this goal do

take place in the photosynthetic membrane. Under low light intensities ( light starva-tion ) the photosynthetic yield will depend upon the efficiency of light energy capture by

antenna pigments and its delivery to the reaction centres. It also relies on the captured

light energy redistribution between photosystem I and II, which operate in series to

form the electron transfer chain from water to NADP. The major fundamental limitation

under elevated light intensities ( light saturation ) arises from differences in the rates of

energy absorption and transfer to the reaction centres of photosystems and subsequent

electron transport. Being much slower than energy transfer, electron transport rates ful-

fil the fundamental thermodynamic requirement; to minimize the uphill reactions and

therefore stabilize energy, which is to be used in the chain of electron/proton transfer

reactions leading to NADPH and ATP synthesis (see Figure 1.1 of Chapter 1).

Maintaining the balanced light energy input into the two photosystems ensures the

optimum working regime of the whole photosynthetic electron transport chain and the

maintenance of the required NADPH/ATP ratio. The photosynthetic light harvesting

antenna efficiently collects light in the shade to feed the reaction centres with energy,

however, an increase in light intensity causes progressive closure of the reaction centres

since they become progressively saturated with energy leading to the reduction of the

fraction of energy utilized in photosynthesis and therefore to the build-up of the

‘unused’ potentially harmful excitation energy in the photosynthetic membrane.

Figure 9.1 summarizes the relationship between the absorbed light energy and the

energy photosynthetic organisms can utilize. The saturation of photosynthetic reactions

with light is inevitable and takes place on its different stages starting from the events

taking place in the photosynthetic membrane, charge separation in the reaction centres,

electron transport and proton gradient formation. Therefore, the light adaptation mech-

anisms are vitally essential to counteract or minimize the previously-described unwanted

effects that undermine the photosynthetic efficiency or even cause the damage to the

photosynthetic membrane.

During evolution plants have developed a multilevel network of adaptation mechanisms

to cope with fluctuations in the light environment (Figure 9.2 ). These can be divided into

the two major groups:

1. Type A adaptations to control light energy input into the photosynthetic membrane, in

other words manage the amount of light absorbed by the antenna pigments (Figure 9.2 ,

managing light absorption );

2. Type B adaptations to deal with the light energy, which has been absorbed by the light

harvesting antenna (Figure 9.2 , managing absorbed energy ).

Rupan_c09.indd 198 8/29/2012 5:35:14 PM

Adaptations of the Photosynthetic Membrane to Light 199

Some type A adaptations work on the level of whole organism. They involve adjust-

ments in the leaf orientation relatively to the direction of incident light. Whilst the

perpendicular orientation will ensure maximum light intensity exposure, nearly-parallel

leaf orientation will greatly minimize it. However, the latter cannot be absolute since there

is always a great deal of light scattered in all directions that is very difficult for the leaf to

avoid. Leaf movements can be of developmental (relatively slow and irreversible), passive

(drought related) and active nature (well-reversible). The latter is based upon the use of a

blue-light absorbing pigments as sensors and the pulvinar motor tissue to drive leaf orien-

tation movements. This adaptive system is very effective in some deep shade-adapted

plants that normally have low photosynthetic rates and are occasionally exposed to high

light. Some desert plants have developed a number of adaptations to increase leaf reflec-

tance and that way they reduce the amount of absorbed light. The A-type adaptations may

include building up inorganic deposits (salt crystals) on the leaf surface or developing

air-filled hairs. As a rule, the efficiency of these protective methods is good but as with the

developmental leaf movements, these adaptations occur on a rather slow timescale.

Type A adaptations occur also on the cellular level. Light absorption can be regulated

by chloroplast movements. This is a relatively fast process, occurring within minutes,

Absorbed energy

Utilised energy

Light intensity

Ene

rgy Excess energy

Low light

High light

Figure 9.1 Light saturation of the photosynthetic energy utilization. In low light the amount of light energy absorbed and the amount utilized in photosynthesis are well-matched. Absorption continues unabated as the light intensity increases but photosynthesis becomes saturated with light. The difference between the amount of light energy absorbed and that utilized in photosynthesis is the ‘excess energy’ which if left unchecked has the potential to harm the photosynthetic membrane.

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but is capable of controlling the light absorption into the photosynthetic membrane only

by 10–20%. The similarity of the action spectra (dependency of the amplitude of

response upon the wavelength of light) for chloroplast and leaf movements suggests that

they are likely to possess common photoreceptors. Factors like the presence of obstacles

such as large vacuoles and other cellular organelles can limit chloroplast movements.

Generally, adaptations on the cell level or at the level of the whole plant have limited

capacity and promptness of response. In addition, scattering of light in natural environ-

ments as well as within the leaf/cell substantially reduces nearly all type A adaptations.

A most profound and fundamental type of plant adaptation to light occurs at the

molecular level. Both, type A and B adaptations take place here. The type A adaptations,

the regulation of light absorption, occur by long-term control of chlorophyll content in the

photosynthetic membrane – regulation of the light harvesting antenna size and the ratio

Plant adaptations to light

Number ofmembranes

Size of antenna

ChloroplastmovementsChloroplast

number

Plantmetabolism

Chloroplastmetabolism

Photosystems ratioState transitions

NPQ

Leaf orientationLeaf reflectance

Salt depositsLeaf structure

Managing light absorption

Managing absorbed energy

Figure 9.2 Multilevel strategies of plant adaptations to the light environment. Type A : managing light absorption; type B : managing absorbed energy. Three levels of adaptations are presented: organismal (plant), cellular (chloroplasts) and molecular (photosynthetic membrane). Reprinted with permission from Ruban, A.V. © 2009, Landes Bioscience. (See Plate 9.2 in colour plate section.)

Rupan_c09.indd 200 8/29/2012 5:35:14 PM


between the number of units of the two photosystems ( photosystems ratio ). The type B,

adaptations that deal with absorbed light, consists of mechanisms that take place in the

photosynthetic membrane and manifest in dynamic changes ( short-term ) in the

fundamental properties (described in Chapter 5) of light harvesting antenna, absorption cross- section and excitation energy lifetime .

9.2 Long-Term Regulation of the Photosystem Ratio and their Antenna Size: Acclimation

There is no strict temporal criterion to distinguish the long-term from the short-term

adaptation mechanisms. Long-term adaptations are predominantly of developmental nature,

and often are a result of regulation of the complex light-dependent gene expression, occur-

ring on transcriptional, translational and post-translational levels. This process takes days

and weeks and on the level of thylakoid membrane involves significant compositional and

structural alterations. These are A-type of adaptations since they tune the photosynthetic

membrane to the optimum absorption of light. They include a well-coordinated modulation

of the composition of the photosynthetic membrane by adjustments in amount of

light-harvesting antennae, PSII and PSI reaction centres, electron transport complexes and

ATPase. Plants growing in limited light possess low photosynthetic rates that cause the

saturation of the amount of utilized energy (the light saturation curve, see Figure 9.1 ) at very

low light intensities. However, plants grown under high light environment possess much

higher photosynthetic capacity revealing saturation at relatively high light intensities. Some

plant species prefer to grow in shade, others in open spaces. However, the extent of long-

term adaptation of the photosynthetic rate can be remarkably broad in some plant species,

showing that they can be equally successful in growing at a broad range of light intensities.

The adaptive changes in PSI and PSII antenna size by light intensity is a well- documented

and highly conserved phenomenon. In plants, grown under high light intensity the antenna

is always smaller than in those grown in shade. It was found that only loosely bound LHCII

complexes are involved in acclimative modulation of the PSII antenna size. The mechanism

of this process involves the reduction in amount of LHCII trimers by the proteolysis of their

apoproteins. This proteolysis is targeted specifically to the loosely-bound complexes. The

protease responsible for the process is suggested to be of serine and/or cysteine type. It is

an extrinsic, stroma exposed thylakoid membrane associated ATP-dependent enzyme.

It takes up to two days for the enzyme expression/post-translational activation after

exposing plants to high light environment. Once activated, it takes less than a day to

complete proteolysis and reduce the LHCII content by 30%. ‘Wasting’ this amount of

LHCII will release about 320 kDa of the protein mass and 160 Chl molecules per PSII

supercomplex. This is significant enough to justify the consideration for their role in the

chloroplast metabolic pathways. In this connection, the possible role for ELIPs was

suggested, as transient carriers of the free chlorophyll released during LHCII breakdown.

The other long-term acclimative response of the thylakoid membrane to the change in

the light environment (quantity and quality) involves alteration in the ratio between

Rupan_c09.indd 201 8/29/2012 5:35:15 PM


number of photosystem I and II units. For example, shade plant, grown under light filtered

on the forest canopy (far red light enriched) will possess higher PSII/PSI ratio to

compensate for less red light, exciting PSII. Although related to the antenna this is

however rather the result of the electron transfer adaptation than light-harvesting. On the

other hand, all these long-term responses have some common features. First of all, the

long-term adaptations are designed to optimize the whole electron transfer shared by two

photosystems. They are also suggested to have some common signal reception pathways

in the acclimation sequence: factor – receptor – transducer – gene . Although the various

aspects of this chain are not well understood, the most recent work on the electron

transport and light harvesting acclimation suggests the redox state of photosystems as

well as the plastoquinone pool to be the important components of the multiple stress

signalling and transducing mechanisms. Recently it has been suggested that the protein

phosphorylation (see the following), including LHCII, can play the signal transduction

role. A one important function of the long-term alterations in the photosynthetic membrane

structure/composition is that they create an optimum molecular environment for the

functioning of the short-term adaptation mechanisms.

9.3 Short-Term Adaptations to Light Quality: State Transitions

9.3.1 The Phenomenology of State Transitions

Normally, during the short-term adaptations no gene expression is involved. They occur

within seconds and minutes and serve to counteract more fast changes in the light

environment, such as diurnal fluctuations in the light quantity and quality, sunflecks, light

filtering by the canopy and so on. All short-term adaptations are of B -type, since they cope

with the light which energy already is absorbed by the photosynthetic membrane. The

most documented short-term process of adaptation to the light quality resulting in the

imbalance of the energy absorbed by photosystems is known as state transitions . This

process occurs within minutes and is effective at low light intensity, that is, when light is

a limiting factor for the functioning of the photosynthetic membrane (see Figure 9.1 ). The

photosynthetic state transitions evolved to balance the excitation energy flows into the

photosystem I and photosystem II reaction centres. It is the most effective in cyanobacte-

ria and green algae, which normally tend to inhabit low light environments. State transi-

tions also take place in the photosynthetic membrane of higher plants. The cause of this

adaptation is simple: PSI and PSII possess spectrally different antenna systems, with PSI

being longer wavelength photosystem than PSII. Figure 9.3 shows room temperature

absorption spectra of isolated PSII membranes and PSI complexes. PSII possess more

chlorophyll b absorption at 650 nm and blue-shifted chlorophyll a with the maximum at

676 nm. PSI has less chlorophyll b absorption and shifted into the far red chlorophyll a

absorption with the maximum at 681 nm and a long ‘tail’ stretching up to 720 nm. Hence,

at low light, it is essential for the both photosystems to receive balanced input of light

energy; otherwize the one which gets less light would limit the efficiency of the whole

electron transport chain. Diurnal changes in the light quality as well as the periodic

Rupan_c09.indd 202 8/29/2012 5:35:15 PM


changes in the canopy shading can strongly alter the balance of red and far-red light expo-

sure of plants grown in low light environments.

Pulse amplitude modulated (PAM) fluorimetry (described in Chapter 4) can be used to

monitor state transitions and obtain the key parameters of this adaptation process.

Figure 9.4 shows a typical trace of induction of the state transitions in leaf. It is essential

in these measurements to use low light intensity for selective excitation of photosystem

I or II in order to keep the majority of reaction centres in the open state. Before applying

spectrally defined light, a saturation pulse is used to measure the level of maximum

fluorescence, Fm, in order to assess the state of photosystem II reaction centres.

A combination of red and far red lights, defined using glass filters, was applied to the leaf

as actinic continuous illumination. It induced a relatively fast fluorescence increase. This

increase was due to reduction of Q A , Immediately after the rise fluorescence declined

rapidly as a result of the light activation of the electron transport carriers that caused

oxidation of the PSII. A steady-state level, F s I was reached that correspond to State I, a

state of relatively balanced electron transfer flow between the photosystems. After this

induction phase, far red light was turned off, causing a marked increase in the fluorescence

level far above Fo to F s I’. This rise indicates that the electron removal from the photosystem

II has been slowed down leading to the additional reduction of PSII (Q A ). Continuing

illumination with the red light induced a gradual fluorescence decrease in the F s I’ level,

lasting for about 15 min and reaching a steady state level F s II’. This is the manifestation of

the transition from State I to State II, tuning the balance of excitation energy towards the

PSI consequently re-oxidizing the plastoquinone pool and PSII, causing a decrease in

fluorescence. Turning the far red light on in this state causes a small drop of F s II’ to F

s II

0.10

0.00

640 650

Red

Far red

660

Wavelength, nm

PSII PSI

PSI–PSII

670 680 690 700 710

Abs

orpt

ion

Figure 9.3 Absorption spectra of isolated PSII-containing membranes (BBY) and PSI complexes prepared by the sucrose gradient separation as described by Ruban et al . ( 2006 ). Difference between PSI and PSII absorption spectra is displayed by hollow symbols.

Rupan_c09.indd 203 8/29/2012 5:35:15 PM


level. The smaller this drop the more completely state transitions compensate for the

spectral differences between photosystems. In extreme case in State II the PSII redox state

becomes almost insensitive to the spectral quality of excited light. Saturation pulse reveals

a decrease in Fm level, associated with the transition into State II. During the next 15 min

of illumination with both PSI and PSII lights a slow increase in the F s II fluorescence level

to F s I takes place. The use of a saturation pulse in the end of this period reveals complete

restoration of the Fm fluorescence level. Now, turning off the far red light again causes the

rise in the Fs level suggesting the restoration of the spectral difference between the

photosystems typical of State II. One can run another cycle of illumination and reveal that

these transients are perfectly reversible.

Several important parameters of the fluorescence transients related to the state transitions can

be calculated. First is a degree of energy imbalance induced by the removal of the far-red light:

0

s sF I F IIB

F

′−= (9.1)

This is somewhat an arbitrary parameter, which depends on the ratio between red and far-

red light intensities. It is useful in tuning the set-up for the measurements as a standard for

optimum light choice. The imbalance parameter can also be used to assess the effect of

antenna composition and structure on the spectral differences between photosystems,

which affect the efficiency of the linear electron transport rate.

Time, min15

State I State II State IIState I

FsI’

FsII’ FsII

FmIFm

qT

FmII

FsIForfr

ononoff

fr

Flu

ores

cenc

e, r

el.

30 45

Figure 9.4 Pulse amplitude modulated chlorophyll fluorescence measurement of the state transitions in Arabidopsis leaf. Fo and Fm are the fluorescence levels corresponding to open and closed PSII reaction centres, respectively. r and fr are red and far-red continuous lights. F s I and F s I’ are the fluorescence levels at State I with and without far-red light on, respectively. F s II and F s II’ are the fluorescence levels at State II with and without far-red light on, respectively. qT is a reduction in Fm level as a result of the transition into State II.

Rupan_c09.indd 204 8/29/2012 5:35:15 PM


The second state transition characteristic is the level of F m fluorescence drop in State II.

It is called qT and calculated as:

m m

m

F I F IIqT

F I=

�

(9.2)

qT is regarded as a fluorescence decline, not a true quenching, since it reflects the decrease

in the PSII antenna size (see the following). This is normally variable from 0 to 0.25.

A third useful parameter for the quantification of the state transitions is referred to as qS :

−′ ′=

−′s

s s

F I F IIqS

F I F IIs

(9.3)

qS indicates how effectively state transitions compensate electron transport imbalance for

changing quality of light. The parameter varies from 0 to 1, where 1 indicates 100%

efficiency in the efficiency of rebalancing of the electron transport rate after the changes in

the spectral quality of light.

A fourth characteristic of state transitions is the rate of F s fluorescence changes. It can

simply be estimated as 1/ t 1/2

, where t 1/2

is the time required to change F s by 50%. In addition,

F s can be fitted with, for example, an exponential function in order to obtain the value of

the rate constant.

9.3.2 The Molecular Mechanism of State Transitions

In order to understand molecular events in the photosynthetic membrane that enable state

transitions observed by PAM fluorimetry one can employ low temperature (77 K) fluores-

cence spectroscopy. The method is useful since the low temperature fluorescence spectrum

of the photosynthetic membrane shows distinct PSI and PSII emission bands allowing

quantifying energy redistribution between them. Figure 9.5 (a) compares spectra of isolated

chloroplasts in states I and II, normalized to the PSII fluorescence region. In State II the

PSI fluorescence band at around 735 nm is strongly enhanced relatively to that in the

State I. The difference State II-minus-State I spectrum closely resembles that of purified

PSI (see Figure 5.19, Chapter 5). The observed enhancement of PSI fluorescence during

the transition into State II appears to be very similar to the change related to grana unstack-

ing where spillover of energy from PSII to PSI occurs. However, electron microscopy did

not reveal any significant changes in stacking during the state transitions. Moreover, whilst

the state transitions did not have any significant effect on the photosystem II intrinsic effi-

ciency, spillover caused a large decrease in Fv/Fm – PSII yield (φ PSII

, see Chapter 4,

Equation 4.17). These fundamental discrepancies have been resolved by the emergence of

a new molecular model explaining the state transitions: the LHCII phosphorylation mecha-

nism that will be described later.

Rupan_c09.indd 205 8/29/2012 5:35:20 PM


The reason why the PSI fluorescence was strongly enhanced in the State II becomes very

clear when one can measure the excitation fluorescence spectrum for the PSI emission at

735 nm (see Chapter 5 for the theoretical and experimental background). Figure 9.5 (b)

shows the difference State II-minus-State I 735 nm excitation fluorescence spectrum. This

Figure 9.5 (a) 77 K fluorescence spectra of isolated Arabidopsis chloroplasts in States I and II. (II–I) is a difference spectrum between State II and State I. Spectra were normalized at 680 nm. (b) 77 K difference excitation spectrum State II-minus-State I (II–I) and reversed transmission spectrum of isolated trimeric LHCII (for the method details see Chapter 4).

2.0

1.8

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0660 680 700

State II

State I

(II–I)

(a)

Wavelength, nm

Flu

ores

cenc

e, r

el.

720 740 760

1.6

1.0

(b)

0.8

0.6

0.4

0.2

0.0450

Flu

ores

cenc

e/(1

-T),

rel

.

500

(II–I)(1-T)LHCII

550

Wavelength, nm

600 650 700

Rupan_c09.indd 206 8/29/2012 5:35:21 PM


difference spectrum reveals the cause of the PSI cross-section increase in the State I. The

spectrum clearly resembles the (1–T) spectrum (reversed transmission) of isolated LHCIIb

complex. Structural features at 435, 660, 670 and 676 nm (chlorophyll a ), and 472 and

650 nm (chlorophyll b ) can be clearly seen in the both types of spectra. The ratio between

chlorophyll a and b in the red and Soret regions indicates that the PSI cross-section is

enhanced in State II due to the coupling of major LHCII complex to this photosystem. No

long-wavelength chlorophyll a forms typical for PSII core complexes (CP47) can be

detected in this experiment, suggesting the absence of spillover, or direct interaction of the

hole of PSII complex with PSI as in the case of grana unstacking. The relative cross-section

increase expressed as a ratio between the area below this difference spectrum and the

excitation fluorescence spectrum of 735 nm band for the State I in the red chlorophyll

absorption region (600–710 nm) was found to be around 25%. Taking into account that PSI

carries 166 chlorophylls 25% of that number would correspond to approximately 42

molecules in additional cross section in the State II. This matches well the number of

chlorophyll molecules in LHCII trimer or three LHCII monomers (42 chlorophylls a + b ).

Assuming the ~20% of PSII antenna decrease as a result of the loss of one LHCII trimer

along with the 25% of the PSI cross-section gain increase in PSI antenna, the total energy

balance change between photosystems will reach 50%. This is a significant alteration that

can compensate for the changes in the light environment, particularly in shade growing

plants, which have larger pool of the peripheral LHCII antenna.

What triggers the energetic coupling between LHCII and PSI in State II observed in the

low-temperature fluorescence measurements? Two major discoveries have been crucial for

a development of the currently accepted state transition model. First is a discovery of

phosphorylation of the chloroplast membrane proteins and second is the enzyme kinase

activation by the redox state of the plastoquinone pool. According to the model, the

imbalance in the energy input into both photosystems is reflected by the redox state of

the plastoquinone pool (see Chapter 3 for the electron transfer pathways in the photosynthetic

membrane), which acts as a governor operating two enzymes, kinase and phosphatase,

which phosphorylate and dephosphorylate some of the LHCII polypeptides, respectively.

The kinase targets threonine or serine residues of the stroma-exposed N-terminus of the

polypeptide. The phosphorylation alters the affinity of a subpopulation of LHCII complexes

to photosystem II causing its detachment and migration towards photosystem I in order to

serve as an additional antenna for this photosystem. The mechanism of Phospho-LHCII detachment from PSII can involve either a generation of a very repulsive negative charge

force brought by phosphate or/and alteration of the conformation of N-terminus (helix

dynamics) upon phosphorylation of LHCII polypeptides. The latter concept, proposed by

Allen (Allen and Forsberg, 2001 ), explores the inherent conformational flexibility of LHC,

discussed previously in this book.

The Figure 9.6 schematically illustrates the state transition mechanism. As a result,

PS II cross-section decreases, whilst that of PSI increases. In addition, since the increase

in the PSI cross section occurs at the expense of chlorophyll b and short-wavelength

forms of chlorophyll a of LHCII complexes it makes PSI antenna less spectrally distin-

guishable from that of the photosystem II facilitating further the balanced redistribution

Rupan_c09.indd 207 8/29/2012 5:35:22 PM


of the excitation energy in the thylakoid membrane. Oxidized plastoquinone acting as

an activator of phosphatase causes removal of the phosphate group from LHCII and

subsequent migration of the LHCII complex towards photosystem II. The LHCII phos-

phorylation model was confirmed by numerous experiments. The effect of redox state

of plastoquinone pool on the kinase has been demonstrated by groups of Allen and

Horton (see the review by Ruban and Johnson, 2009 ). The role of the spectral quality of

light in the phosphorylation of thylakoid proteins has been shown on both, isolated

photosynthetic membranes and leaves. Chlorophyll fluorescence measurements have

confirmed reversible, phosphorylation-related alteration in the energy redistribution

between photosystems, resulting in decrease in PSII and increase in PSI electron trans-

port rates. Various microscopy and biochemistry experiments proved the migration of

phosphorylated LHCII into the stroma lamellae, which carry PSI. It was proposed that

only a subpopulation of the major LHCII, a mobile phosphorylated complex (most

likely L- or M-trimers), migrated towards PSI. This complex was found to be enriched

in the Lhcb2 polypeptide.

Loosely-bound LHCII protein phosphorylation provides molecular mechanism for the

state transitions. However, apart from loosely, there are some medium- and strongly-bound

and minor antenna LHCII complexes as well as D1 and others, which undergo phospho-

rylation under some conditions in the photosynthetic membrane and role of this multiple

phosphorylation in the state transitions is not clearly understood. Phosphorylation may, for

example, be a signal to control the thylakoid membrane energization. The phosphorylation

of CP29 complex was suggested to play an important role in the cold stress adaptation of

the membrane. Clearly, the phosphorylation of the thylakoid proteins is an important

Figure 9.6 The LHCII phosphorylation model of the state transitions. Imbalance in the light energy input into the antennae of photosystems leading to the preferential excitation of PSII causes the reduction of the plastoquinone pool (PQH2). Reduced PQ activates kinase, which phosphorylates polypeptides of LHCII. The phosphorylated LHCII detaches from PSII, migrates towards PSI and incorporates into its antenna system. As a result of this incorporation PSI gains more excitation energy which leads to the increase in the reaction centre (RCI) turnover with the subsequent oxidation of the plastoquinone pool. When PSI gains relatively more excitation light than PSII it oxidizes PQ further. PQ activates phosphatase, an enzyme which dephosphorylates LHCII attached to the PSII. Dephosphorylated LHCII detaches from PSI, and migrates and incorporates into PSI. The different colours of PSI (black) and PSII (light grey) antennae highlight their spectral difference.

Rupan_c09.indd 208 8/29/2012 5:35:22 PM


phenomenon and should be widely investigated with respect of the signal transduction,

protein conformation, membrane dynamics and the adaptation mechanisms involved.

One mechanistic question deserving a particular focus is what is the oligomeric state of

the migrating phospho-LHCII complex, a trimer or a monomer ? It has been previously

suggested that the LHCII phosphorylation causes its monomerization, detachment from

PSII and migration towards PSI in the monomeric state. Migration of a smaller, monomeric

complex with an altered conformation allowing recognition of PSI is an elegant scenario

explaining the promptness, precision and effective reversibility of the state transitions.

However, recently, electron microscopic evidence has emerged suggesting that in State II

PSI interacts with a particle closely resembling an LHCII trimer. Figure 9.7 (a) shows the

schematic representation of the result obtained by the Boekema’s group (Kouril et al ., 2005 ) showing a single PSI complex interacting with the single LHCII trimer. This

presentation illustrates the data obtained using the electron microscopy combined the

single particle analysis (see Chapter 4 for the method details) on the digitonin-solubilized

membranes isolated from plants where the State II transition was induced by light.

A docking site around a domain of PSI subunits A, H, I, K and L has been proposed for

LHCII. The binding was interpreted as weak and non-specific.

In addition to the electron microscopy analysis, the question about the oligomerization state

of migrating phospho-LHCII was addressed by analysing the Soret region of the calculated

additional cross-section spectrum of PSI in State II. Figure 9.7 (b) displays the Soret band of

this spectrum and its second derivative as well as excitation fluorescence spectra for the

LHCIIb in trimeric and monomeric states. The second derivative spectrum shows maxima

corresponding to chlorophylls and xanthophylls (see Chapter 8 for details). A band at 510 nm

corresponding to Lutein 621 (Lutein 2) absorption is clearly seen. This band is present

exclusively in LHCII in the trimeric, not monomeric state as evident from the comparison of

their fluorescence excitation spectra. If the phospho-LHCII interacting with PSI would exist

in the monomeric state the cross-section increase spectrum would not show 510 nm band.

Therefore, the excitation spectral analysis of the state transitions on isolated chloroplasts made

it possible to not only identify and quantify LHCII interaction with PSI during the state

transitions but also helped to define in situ its state of oligomerization as a trimer.

9.3.3 Chromatic Adaptations in Plants Lacking the Polypeptides of the Major LHC II Complex

The significance of keeping a balance of excitation energy input into photosystems was

highlighted by experiments on Lhcb2 antisense plants ( as Lhcb2) lacking the two major

LHCII polypeptides, Lhcb1 and 2 (see Chapters 7 and 8 for an introduction to Lhcb2

antisense plants). Figure 9.8 shows a typical PAM fluorimetry of the state transitions in the

antisense leaf (for details of the method see the Figure 9.4 legend and the text). The state

transitions were completely inhibited (qT = 0, qS = 0), as was anticipated since the polypeptide

that undergoes phosphorylation, Lhcb2, was missing in these plants. What was surprizing is

the fact that the imbalance parameter, IB, was found to be drastically reduced in comparison

Rupan_c09.indd 209 8/29/2012 5:35:22 PM


to the wild-type plants (IB as = 0.1 vs. IB wt = 0.65). Indeed, the response to the far-red light

in the antisense plants was much smaller than that of the wild-type (Figure 9.4 ). This indi-

cates some alterations in the photosystems antennae composition/structure that reduced the

spectral differences between them (see Figure 9.3 ). Further experiments have revealed that

significant compensatory adjustments took place in the photosystem II antenna of as Lhcb2.

Figure 9.7 (a) Scheme illustrating interaction of the phospho-LHCII trimer with PSI supercomplex based upon the electron microscopy and single particle analysis work of Kouril and coworkers (2005). Presented in freeware PyMol 0.99. (b) The Soret band 77 K F735 excitation fluorescence spectral difference, State II-minus-State I (II–I), and its second derivative (2nd derivative). The solid and dashed lines represent excitation fluorescence spectra of isolated LHCII trimers and monomers, respectively (detection wavelength was 680 nm). Vertical line highlights the 510 nm band characteristic for the LHCII trimer and absent in the complex in the monomeric state.

(a)

Lhca1

Lhca4

Lhca2Lhca3

Phospho-LHCII

(b)

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

420 440

(II–I) Monomer510

Trimer

(II–I) 2nd derivative

460 480

Wavelength, nm

Flu

ores

cenc

e, r

el.

500 520 540–0.2

Rupan_c09.indd 210 8/29/2012 5:35:22 PM


The major LHCII trimers were still present in the antisense pants but built of Lhcb5 and

Lhcb3 proteins. Close comparison of the spectral properties of these trimers and the timers

isolated from the wild-type revealed not only a significant decrease in the chlorophyll b

absorption but also a shift towards longer wavelengths of the spectrum of chlorophyll a

(Figure 9.9 a). These differences made PSII antenna of antisense plants effective in capturing

less red and more far red photons and therefore made it similar to the PSI antenna.

Furthermore, a significant increase in the PSI antenna size caused by an increased amount of

LHCI complexes took place. This was a first observation that LHCI antenna can be consider-

ably upregulated. The excitation fluorescence spectroscopy of isolated PSI revealed the

functional increase in cross-section by approximately 20% (Figure 9.9 b). However, this fig-

ure is probably much higher taking into account the removal of a large proportion of the

peripheral LHCI from PSI during its purification in the mutant. PSI antenna in the antisense

plants gained more chlorophyll b and blue-shifted chlorophyll a with additional LHCI units

and therefore became more blue-shifted: the response completely opposite to that of the PSII

antenna, as described previously. Therefore, the spectral alterations in antennae of photosys-

tems in the Lhcb2 antisense plants significantly decreased chromatic differences between

the two photosystems. This makes the photosynthetic membrane less sensitive to the changes

in spectral quality of light as was clearly indicated by the dramatically decreased imbalance

parameter in the state transition measurements shown in Figure 9.8 . Absence of ability to

perform the state transitions seems to be compensated by the decrease in the spectral differ-

ences between PSI and II antennae in as Lhcb2 plants. Alleviation of these differences is yet

another demonstration of how flexibly the photosynthetic antenna can respond to dramatic

alterations in the polypeptide composition ensuring the principle of robustness .

Figure 9.10 illustrates the antenna adjustments in the as Lhcb2 plants. The state transi-

tions are absent and compensated by the decreased PSII antenna (compare to the Figure 9.6 ),

which is less spectrally different from the PSI antenna (highlighted by the same grey colour

of the antennae of the both photosystems). Photosystem I antenna is increased with more

LHCI units attached.

Flu

ores

cenc

e, r

el.

rfr fr Off On

5 min

Figure 9.8 Absence of the state transitions in Lhcb2 antisense Arabidopsis leaf. For experimental details, see Figure 9.4 .

Rupan_c09.indd 211 8/29/2012 5:35:23 PM


9.3.4 Future of State Transitions Research

State transitions remain one of the most interesting topics in photosynthesis research.

Invention of new experimental approaches is likely to ignite a new wave of discoveries in

this field. Development of various microscopies, suitable for studies of the membrane

structure and dynamics in vivo can be very promising in elucidating the mechanism of the

phospho -LHCII delivery to the PSI; that is, migration character, rates, role of the membrane

composition and macrostructure, character of interaction of the phospho -LHCII with PSI.

Availability of various protein and pigment (xanthophyll) antenna mutants provides a rich

Figure 9.9 (a) 77 K absorption spectra of isolated LHCII trimers from the wild-type (solid line) and Lhcb2 antisense (dashed line) Arabidopsis leaves. (b) 77 K reversed transmission (solid lines) and F735 fluorescence excitation (dashed lines) spectra of isolated PSI from the wild-type (wt) and Lhcb2 antisense ( as ) plants.

(a)

0.25

0.20

0.15

0.10

0.05

0.00640

wt

as

660

Wavelength, nm

Abs

orpt

ion

680 700

(b)

1.0

0.8

0.6

0.4

0.2

0.0640

as

as

wt

wt(1-T)

Excitation

1-T

/Flu

ores

cenc

e, r

el.

660

Wavelength, nm

680 700 720

Rupan_c09.indd 212 8/29/2012 5:35:24 PM


field for experimentation, which could help to determine what factors govern detachment,

migration and energetic coupling to PSI of the phospho -LHCII and roles of the immobile

PSII antenna complexes and LHCI in the state transitions. Important physiological

questions regarding the state transitions are waiting to be addressed. Among them are

possible effect of LHCII antenna size and composition; effect of membrane fluidity and

protein density; effect of the xanthophyll cycle activity; effect of PsbS; effect of the change

in the photosystem stoichiometry and PSII connectivity; the role of state transitions in plant

survival under the light starvation regimes in natural environments. These and other new

approaches should engender a greater understanding of the role of the state transitions in

the context of the fundamental adaptive responses of the photosynthetic membrane and

indeed the life of the plant as a whole.

One important aspect of the state transitions and LHCII phosphorylation is interaction

with the long-term adaptation mechanisms, their role in the cyclic electron transport and

interaction with the other short-term adaptation mechanisms. As mentioned previously, the

state transitions should be particularly effective under low light, where the thylakoid

membrane accumulates large PSII antenna and, basically due to the presence of the loosely-

bound LHCII. This population is also a substrate for phosphorylation, therefore in low-light

grown plants the extent of the state transitions or phosphorylation-induced energy

redistribution between photosystems is the highest. It can be rationalized that the shade-

grown plants are appropriately better prepared for sudden fluctuations in the light quality,

which can originate from the sunflecks and changes in shading from the other plants

(movements due to wind, diurnal change in the angle of sun beams etc.). On the other hand,

in plants grown under high, saturating light (Figure 9.1 ), where state transitions lose their

role, the loosely-bound LHCII antenna is reduced. Therefore, large peripheral LHCII

antenna along with the long-term increase in PSII/PSI ratio (see previous) will constitute

an adequate basis for more flexible adaptation to the sudden energy imbalance between

photosystems. Combining ecophysiological experiments with some genetic manipulation

approaches, designed to specifically under- or overexpress certain components of

the thylakoid membrane seems to be a way forward towards shedding more light upon the

Figure 9.10 The model illustrating compensatory adjustments in both photosystem I and II antennae as a response to the lack of Lhcb1 and 2 polypeptides of the major LHCII complex. Lack of the state transitions is compensated by the reduced PSII and increased PSI antenna sizes. In addition, in the as Lhcb2 plants, PSII antenna absorption is red-shifted in comparison to that of the wild-type plants. PSI antenna absorption, on the other hand, shifted more to the blue, since it is more enriched in chlorophyll b -containing LHCI complexes. Similarity of PSI and PSII antennae colouring (both in grey) indicates their similar spectral properties (compare with the Figure 9.6 ).

Rupan_c09.indd 213 8/29/2012 5:35:24 PM


molecular mechanism and significance of the state transitions and protein phosphorylation

in the natural plant habitats.

9.4 Short-Term Adaptations to Light Quantity

In high light, the energy balance between photosystems is not a problem. However, at

increasing light intensity the photosynthetic reaction centres become progressively

saturated (closed), resulting in a reduction in the fraction of energy utilized in photosynthesis

and the subsequent build-up of ‘unused’, potentially harmful, excitation energy in the

photosynthetic membrane (Figure 9.1 ). Build-up of this energy can cause various

detrimental effects on the organism, particularly on the delicate photosynthetic machinery.

The excess excitation energy in the antenna systems can cause photoinhibition , a sustained

decline in the photosynthetic efficiency and productivity, associated with the damage of the

photosynthetic reaction centres. The reaction centre of the PSII is more susceptible to

the damage than the reaction centre of PSI because of the very strong oxidation potential

of the P680 (+1,17 V) needed to oxidize water (see Chapter 3). Under some conditions,

when electron donation to P680 is less efficient than oxidation, an increase in the P680 +

lifetime will occur. The powerful oxidant P680 + will inevitably oxidize nearest pigments

and aminoacids, causing their degradation and the subsequent D1 degradation will follow.

This type of damage is called donor side photoinhibition . In other circumstances, when

acceptor side is less efficient, a radical pair is formed. The recombination of this pair will

lead to the P680 triplet formation. In this state P680 can interact with the atmospheric

triplet oxygen, causing the formation of very reactive singlet oxygen, which in turn will

bleach P680 leading to the degradation of D1. This type of photodamage is called acceptor side photoinhibition Therefore, regardless of the damage scenario, the number of active

PSII units will be decreased and because of the slow D1 repair the decline in electron trans-

fer will last for some time even when excess light is no longer present. The excess energy

accumulation is very dangerous for wild and particularly openly cultivated plants, exposed

to full sunlight. This is becoming an important problem for agriculture; the cost of

photodamage is very high. The need to avoid photoinhibition has therefore created a strong

selective pressure for mechanisms that reduce excess energy accumulation in high light

environments. Therefore the study of molecular mechanisms of photoprotection is crucial

and the rest of this review will deal with them. These are fundamental, molecular

mechanisms designed by evolutionary process to supply the basis for the photosynthetic

light utilization flexibility, regardless the place of plant habitat, some of them are very

effective and prompt responses, achieved within minutes or even seconds.

9.4.1 Control of Excess Light Energy in Photosystem II – The Phenomenon of Nonphotochemical Chlorophyll Fluorescence Quenching (NPQ)

Excess light energy in the photosynthetic membrane generated under high illumination

conditions can be controlled. This adaptation mechanism emerges from the very heart of

Rupan_c09.indd 214 8/29/2012 5:35:24 PM


the energy transformation events depicted and explained in Figure 1.1 of Chapter 1, taking

place in the photosynthetic membrane. The scheme shown in Figure 9.11 illustrates the

fundamental principle of the light harvesting and, hence, the excess energy control in the

photosynthetic membrane. The excess light have to be controlled over the sequence of

energy transformation events in the photosynthetic membrane. In the light phase of photo-

synthesis, overproduction of ATP (NADPH) will cause the accumulation of protons in the

intrathylakoid membrane space ( lumen , see Chapter 3), which in turn will lead to inhibition

of a number of key electron transport enzymes (Cyt b/f and the oxygen evolving complex

of PSII) causing reduction in electron transport rates. Indeed, as with respiratory control in

mitochondria, the photosynthetic membrane possesses photosynthetic control as a feedback

mechanism for balancing ATP (NADPH) production with electron transport. However, no

matter how efficient photosynthetic control is, the path of photon to electron energy conver-

sion requires an additional control loop. This requirement is based on the fact that the light

capture and delivery of energy to reaction centres by antenna is a very efficient process that

is not directly limited by the reaction centre turnover rate. The essential feedback control exists in the form of the lumen proton effect upon the PSII antenna (Figure 9.11 ). Protonation

of some aminoacid residues of PSII light harvesting complexes causes the formation of an

additional nonradiative energy dissipation channel in PSII that competes with reaction

centres for excitation. This competition leads to downregulation of antenna efficiency that

relieves the photosynthetic membrane from excess light. This feedback process can be

monitored in the form of the PSII antenna chlorophyll fluorescence yield decline under

conditions of excess excitation energy accumulation in the photosynthetic membrane and is

called nonphotochemical chlorophyll fluorescence quenching, qN, or NPQ.

Chapter 4 has already introduced qN with the PAM fluorescence analysis technique,

explaining how qN/NPQ parameters are calculated and illustrated the time course typi-

cal of the short-term adaptation that is reversible and occurs within the minutes time

scale (see Figure 4.15). Figure 9.12 shows parallel monitoring of chlorophyll fluores-

cence by PAM and 9-aminoacrydine fluorescence of isolated chloroplasts. The latter is

used to measure the level of the proton gradient across the photosynthetic membrane,

ΔpH (described in Chapter 4). The ΔpH establishment is induced by actinic light (AL)

and causes the decline in the fluorescence level of 9-aminoacrydine. The recording

shows that ΔpH forms faster than chlorophyll fluorescence quenching, qN. It takes

Photosystem II E.T.

NPQ

ATP

NADPH

ΔpH

Figure 9.11 Scheme depicting the feedback control of light harvesting in plants. PSII absorbs light and produces electrons (ET) which are transported through the thylakoid membrane to produce NADPH and drive formation of Δ pH for ATP synthesis. Built up Δ pH exerts control over PSII via regulation of the excitation pressure in LHCII antenna (NPQ).

Rupan_c09.indd 215 8/29/2012 5:35:24 PM


about 10 s to reach maximum for ΔpH whilst quenching of Fm’ (the onset of qN) takes

hundreds of seconds (several minutes). After about 5 min an inhibitor of PSII electron

transport (DCMU: blocks plastoquinone (PQ) binding site QB) prevents electrons leav-

ing PSII and causes complete reduction of Q A acceptor. The latter ‘closes’ reaction cen-

tre and prompts chlorophyll fluorescence rise to the maximum. As a result, the whole

electron transport of the photosynthetic membrane stops causing the collapse of ΔpH

that is registered by recovery of 9-aminoacrydine quenching within ~10 s. The chloro-

phyll fluorescence quenching, qN, also recovers, but on a slower time scale and not

always to the initial level, Fm. If the Fm“ < Fm, this indicates the onset of photoinhibi-

tion or some slowly reversible components of qN that are related to photoprotection (see

following). The quickly reversible component of qN, qE, is normally used as a measure

of the short-term photoprotective response of the photosynthetic membrane to the high

light exposure. Figure 9.13 demonstrates how important qE is for the protection of PSII.

The yield of PSII is measured with and without qE: the uncoupling agent, nigericin,

prevents formation of ΔpH and hence qE. The excess light illumination for ~1 h caused

a strong irreversible reduction in the PSII yield (Fv/Fm) from 0.8 to <0.4. This means

that within one hour moderate illumination destroyed more than 50% of PSII reaction

centres. The yield recovery will take place on the timescale of hours since it requires

replacement of the damaged reaction centre components, mainly the D1 protein, by a

newly-synthesized one. The long-term damage to the RCII undermines overall plant

growth and development.

Figure 9.12 Simultaneous measurements of ∆pH (9-aminoacrydine fluorescence quenching) and chlorophyll fluorescence quenching induced by actinic light (AL, 1000 m Mm −2 s −1 ) illumination. Chlorophyll a fluorescence levels at the conditions of all open (Fo) or closed (Fm) RCII, steady-state at actinic light (Fs), quenched by NPQ (Fm’) and recovered by the inhibitor of PSII, DCMU (Fm”) are indicated.

Rupan_c09.indd 216 8/29/2012 5:35:25 PM


The differences in kinetics of ΔpH and qN indicate that the processes are not entirely

tightly interconnected. The ΔpH effect on qN is considered as a trigger type, which only

initiates or ignites the process of quenching formation within the PSII antenna: a quenching

site . Antenna response to the trigger is somewhat delayed, suggesting that there are other

events that should happen at the site in order to form qN. Hence, a change in the site is

anticipated, that forms a quencher , that begins to compete with RCII for the excitation and

therefore lowers the excess energy in the photosynthetic membrane, protecting it from the

photodamage. Trigger , site , change and quencher are four key elements that constitute the

molecular mechanism of qN. The term NPQ is now more often used to refer to the nonpho-

tochemical quenching in general and its photoprotective component, qE, that is often called

photoprotective NPQ. Both terms, qE and protective NPQ , are equally appropriate to name

the photoprotective component of qN in general. As far as the calculation methods used,

qE-type is usually the yield of the process (Fm″ - Fm′)/Fm″, whilst protective NPQ is

Fm/Fm′ - Fm/Fm″ and characterizes the power of the process relatively to the sum of the

rates of other dissipative channels in antenna: k D /(k

f + k

d ) (see Chapter 4 for details). In

most cases Fm“ < Fm, meaning the dark recovery of NPQ is never complete. Later we will

discuss the multiple factors that prevent this recovery.

9.4.2 The Molecular Components and Processes Involved in NPQ

To understand the molecular mechanism of NPQ requires identification of proton-interacting

complex(es) of antenna and their aminoacids, identification of effect(s) on the structure

0.8

0.6

0.4

0.2

0.00 20 40 60

Time, min

+ Uncoupler

Control

ΦP

SII

Photoinhibition

80 100 120

Figure 9.13 The photoprotective effect of NPQ. The actinic light was switched on at the zero time on the time scale. Following ~1 h illumination PSII yield was promptly restored in the dark to normal, ~80%, indicating that the light did not cause any sustained photoinhibitory damage. However, when Δ pH was inhibited with the uncoupler nigericin to prevent the formation of NPQ, a significant sustained decrease in PSII yield took place following illumination, indicating the onset of the photoinhibition (highlighted by the down arrow).

Rupan_c09.indd 217 8/29/2012 5:35:26 PM


protons inflict as well as finding out of identity and the process of activation of trap(s) of

the excess energy in antenna. As shown in Figure 9.12 , acidification of the thylakoid lumen

is crucial for NPQ. It is possible that some lumen-exposed or closely-localized to the lumen

site aminoacid residues, mainly aspartates and glutamates, of LHCII complexes are

involved in the process of proton binding. The research is underway to link NPQ to the

protonation of these residues. It is not entirely clear whether the protonation process is

specific or is of generic character that involves multiple proton-binding sites. The question

of protonation inevitably addresses the question of the site, which antenna complex (or

indeed complexes) is/are involved in NPQ? The rest of this chapter will present a current

mechanistic understanding of the molecular events underlying photoprotective NPQ (qE).

9.4.2.1 The Site of qE

The knowledge that qE occurs in the light harvesting antenna came from several spectro-

scopic and biochemical studies:

1. qE was found to be associated with a significant decrease in LHCII fluorescence when

all PSII reaction centres are open (Fo quenching);

2. qE was discovered to persist if samples were frozen to 77 K;

3. It was associated with quenched fluorescence bands originating from LHCII com-

plexes. Spectral analysis of qE and qP showed that different emitting bands are

quenched: qP preferentially quenches the PSII core fluorescence (685 and 693 nm

bands) and qE quenches 680 and 700 nm bands that belong to LHCII;

4. The time-resolved fluorescence recorded for leaves was consistent with quenching

taking place in the antenna;

5. Direct measurement of heat emission in the qE state showed it to occur promptly that

can only happen in antenna complexes;

6. Mutant plants lacking the most of LHCII antenna possess much reduced levels of qE;

7. Cross-linkers that bind to protein blocked qE in the same way as restricted the transi-

tion of isolated LHCII from efficient to protective states (see Chapter 8);

8. qE and isolated trimeric LHCII responded in the same way to a number of factors such

as pH, antimycin A, dicyclohexylcarbodiimide (DCCD), tertiary amines and magne-

sium;

9. qE was found to be almost entirely dependent upon the presence of exclusively LHCII

antenna-bound xanthophylls, lutein and zeaxanthin;

10. The kinetics of qE were greatly dependent upon the presence of PsbS protein that is

not a part of PSII core complex and is rather relatively randomly redistributed in the

photosynthetic membrane.

Summarizing the facts listed above on the origins of qE one can conclude that the process

occurs in the light harvesting antenna and depends apart from the trigger , ΔpH, upon a few

factors: zeaxanthin (lutein), PsbS protein and the state of LHC aggregation. All these fac-

tors will be discussed in the following sections in order to gradually build the picture of our

current understanding of the molecular mechanism of qE and other NPQ components.

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9.4.2.2 The Role of Zeaxanthin in qE

Demmig-Adams et al . (1990) made one of the major breakthroughs towards understanding

the mechanism of qE. This group provided the first evidence of a connection between the

xanthophyll cycle and NPQ before the details of PSII antenna composition and structure

have been revealed. The xanthophyll cycle was discovered by Sapozhnikov in 1957 and its

properties and enzymatics were initially characterized by Yamamoto and Hager (Yamamoto

et al ., 1962 ; Hager, 1966 ). The cycle involves two enzymes, the de-epoxidase and the

epoxidase, which reversibly interconvert violaxanthin and zeaxanthin (see Figure 5.14 for

xanthophylls’ structure comparison). In leaves it takes minutes to induce the synthesis of

zeaxanthin from violaxanthin but hours for the back reaction. Therefore, zeaxanthin accu-

mulated in light remains present in the dark for some time, when qE is absent. The work

of Demmig-Adams showed that the conversion of violaxanthin into zeaxanthin, induced

by the formation of Δ pH in high light, strongly enhanced NPQ. They suggested that zeax-

anthin may be the pigment responsible for quenching (see the following for a further

discussion of the origins of NPQ quenchers). Figure 9.14 shows two PAM fluorescence

induction curves obtained on a spinach leaf induced by two consecutive illumination peri-

ods of 5 min each interrupted by 10 min darkness. This is a method of application of cycles

of light and darkness in order to see the photosynthetic membrane response and capacity

to form NPQ. Indeed, in the natural environments light exposure can be intermittent; hence

Figure 9.14 Influence of zeaxanthin upon NPQ formation in leaves. All fluorescence induction terms as in Figure 9.12 . The Fm level dark recovery after the first illumination cycle (Fm") is indicated by a vertical arrow. qI is the amount of irreversible quenching after 10 min darkness. qN – Zea is the amount of quenched Fm in the absence of zeaxanthin. qN + Zea is the amount of quenched Fm promoted by the presence of zeaxanthin.

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the experiment shown in Figure 9.14 is aimed to model it. The leaf was dark-adapted and

did not contain any zeaxanthin or antheraxanthin (an intermediate containing only one

epoxy group, hence half molecule is violaxanthin and half is zeaxanthin). In the first 3 min

of the first illumination cycle qN formed in the total absence of zeaxanthin (qN -Zea

) but was

only about 0.4 or when expressed as NPQ – ~0.67. After the third illumination minute qN

increased further starting the second, slower formation phase that correlated with emer-

gence of zeaxanthin (see the inclined arrow). The Fm ’ quenching was followed by the

quenching of Fo. Indeed, after turning off actinic light, the Fo ’ level was registered slightly

lower than Fo. After 10 min in the dark the major part of qN has recovered (qE). The

remaining quenching corresponded to qI and was about 0.2. The PSII yield, φ PSII

or Fv/Fm

was reduced from 0.8 to about 0.7. This reduction is significant and could be caused either

by photoinhibition ( damage of some RCII) or the presence of sustained photoprotective

NPQ. The second illumination cycle revealed three very important features of NPQ

( summarized in Figure 9.15 ):

1. the amount of ΔpH-triggered qN is much higher in the presence of zeaxanthin reaching

70% that corresponds to NPQ ~2.3. Hence, the power of NPQ in the presence of zeax-

anthin rose by more than three times;

2. the quenching was almost 100% reversible, indicating that the amount of qI was much

lower compared to that induced in the first illumination cycle;

3. it formed much faster (but relaxed much slower) than in the first illumination cycle.

The three described features of the NPQ behaviour during the second illumination cycle

suggest that NPQ only partially depends upon zeaxanthin but is greatly enhanced in its

presence. In addition in this experiment qI was largely dependent upon zeaxanthin-related

NPQ component implying its rather photoprotective and not photoinhibitory nature. Further

investigation of zeaxanthin-dependent qI revealed that it consists of two components, one is

Figure 9.15 Maximum fluorescence levels in the dark (Fm) and after 5 min illumination (∆pH) with violaxanthin or zeaxanthin (Fm ’ ). Zeaxanthin slowed down the dark recovery of Fm ’ (for details see the Figure 9.14 legend).

Rupan_c09.indd 220 8/29/2012 5:35:26 PM


sensitive to uncouplers and the other is not. Simultaneous measurements of Δ pH and NPQ

revealed that the uncoupler-sensitive component of zeaxanthin-dependent qI persists for

relatively long periods in darkness and is not associated with the bulk Δ pH. Therefore, a

new aspect of photoprotective quenching in the antenna was revealed: namely that the lon-

gevity of the photoprotective state in the darkness could vary depending upon zeaxanthin

concentration and pre-illumination history. The modulation of qI by zeaxanthin led to the

possibility that it may originate from the same site and the same process that underlies qE.

It was found, for example, that conditions promoting the zeaxanthin-dependent qI compo-

nent caused a concomitant decrease in qE, as if qE was in fact becoming less and less

reversible. This idea is consistent with the observed acceleration of NPQ formation coupled

to strong deceleration of its recovery induced by the presence of zeaxanthin. An extreme of

such behaviour of NPQ when it became gradually totally irreversible even after addition of

an uncoupler, was discovered in a diatom alga Phaeodactylum and also in plants exposed to

high light and low temperature conditions. Summarizing the observations on the irreversible

in the dark component of NPQ we can conclude that its nature is complex and it consists at

least of the following sum: qI + qZ + qH + , where qI is a photoinhibition-related component;

qZ is the zeaxanthin-dependent and ΔpH-independent component and qH + is the uncoupler-

sensitive component promoted by the presence of zeaxanthin.

Observations of the relationship between NPQ and the xanthophyll cycle activity

described here found some explanations in the biochemical and spectroscopic studies on

PSII light harvesting antenna described in Chapters 6–8. This work revealed that all

xanthophyll cycle carotenoids of PSII are associated with LHCII antenna. Almost all of

them are bound to the major LHCII complex peripheral site V1. Their affinity of binding to

this site is much lower than in the rest of xanthophylls, therefore they are easily accessible

for the enzymes of the xanthophyll cycle. Most strikingly, in vitro fluorescence quenching

experiments on isolated LHCII showed very strong amplification effect of zeaxanthin

(Figure 8.20, Chapter 8). pH titrations of the quenching induced in isolated LHCII and the

ΔpH titration profiles of qE in isolated chloroplasts in the presence of violaxanthin or zeax-

anthin revealed similarities. Figure 9.16 shows these pH-dependencies. In both, isolated

LHCII and chloroplasts zeaxanthin affected the quenching in such a way that it became

more responsive/sensitive to acidification. Whilst with violaxanthin pK for protonation was

within 5.5 for the both, in vivo and in vitro experiments, zeaxanthin shifted it to 6.5–6.8 by

>1 pH unit. This is a remarkable manifestation of a control of NPQ by the xanthophyll cycle.

The mechanism of this control is based upon the alteration of LHCII antenna sensitivity to

the lumen protons. Interestingly, in the absence of zeaxanthin under somewhat low lumen

pH conditions the amount of nonphotochemical quenching becomes very similar to that in

the presence of zeaxanthin. This indicates that zeaxanthin is not indispensable for NPQ.

There are two specific features in the pH dependency of the quenching in vivo . First, is that

the xanthophyll cycle seems to have stronger effect here in comparison to that on isolated

LHCII. Indeed, the + Vio and + Zea titration curves are further apart for chloroplasts in

comparison to those of LHCII. Second, for in vivo case the shape of titration curves with

violaxanthin and with zeaxanthin differs. For violaxanthin-containing chloroplasts the shape

is sigmoidal , whereas for zeaxanthin-containing chloroplasts it is rather hyperbolic . This

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behaviour is indicative of the presence of allosteric control in the activation of qE. Similar

effect was also observed on isolated LHCII complexes but to a lesser extent. pH titration

experiments enabled Horton and coworkers (Ruban and Horton, 1999 ) to suggest that the

xanthophyll cycle plays a regulatory role in qE with zeaxanthin acting upon LHCII as an

allosteric activator, not a direct chlorophyll excitation quencher, by making the complex more

sensitive to pH, in other words promoting the proton-antenna association constant, pK. Later,

the model suggesting the molecular mechanism underlying this effect on the level of the single

LHCII monomer will be presented. The allosteric model of qE explained reduced levels of

quenching observed in dark-adapted leaves ( up-arrow , Figure 9.16 ) and enhanced levels of qE

in the second illumination cycle and, most importantly, much slower recovery of the quenching

in the presence of zeaxanthin. Indeed, as indicated in Figure 9.16 , in the darkness followed by

the second illumination cycle ( down-arrow ) zeaxanthin-enhanced antenna sensitivity to

protons would inevitably lead to the reduction in recovery rate since qE would still be present

at relatively high levels of lumen pH. The differences in lumen pH sensitivity of the photo-

synthetic antenna during and after illumination caused by the xanthophyll cycle activity

manifest a hysteresis in PSII adaptation to high light. Hysteresis provides a mechanistic base

for the photosynthetic membrane memory to illumination. In low light, the amount of zeaxan-

thin will be very low making NPQ not very sensitive to ΔpH. Brief exposure to a moderately

high light will induce qE of somewhat low amplitude. The levels of photoprotection and tran-

sient PSII yield decline will be low. The light harvesting antenna will be geared for efficient

collection of light photons and PSII yield will be high on average. Increase in the ambient light

4

Chloroplasts

Flu

ores

cenc

e qu

ench

ing

Chloroplasts

LHCII

+Zea

+Vio

65

pH

7

Figure 9.16 Effect of the xanthophyll cycle carotenoids on the nonphotochemical quenching pH-titration curves. Solid lines : NPQ in vivo, lumen pH was estimated using the 9-aminoacrydine method. Arrows indicate the relationships between NPQ and lumen pH at the conditions of dark-adapted state with violaxanthin (up arrow) and after induction of zeaxanthin formation (down arrow). Dashed lines : pH titrations of fluorescence quenching in isolated LHCII trimers.

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intensity, frequency and duration of exposure would cause gradual accumulation of zeaxan-

thin. The photosynthetic antenna would gradually become sensitized to light, the levels of

attained NPQ would become higher and the quenching recovery in the low light would become

slower. This would lead to conditioning of the photosynthetic membrane to a photoprotective

mode that would manifest in more frequent and even sustained accumulation of nonphoto-

chemical quenchers in light harvesting antenna leading to the decline in the PSII yield. The

hysteresis in PSII is therefore a way to economically harvest light and enable the most efficient

and safe photosynthesis in chloroplast membranes. In addition, temporal separation of the

trigger (ΔpH) effect and the change in antenna that produces qE makes the membrane response

to light smooth, avoiding frequent dramatic alterations in the electron transport rates and

generation of NADPH and ATP. Indeed, as far as the light is not of a dangerous intensity and

duration it is being efficiently used by PSII with its effective antenna. When the light begins

to threaten the reaction centres, the antenna becomes gradually inefficient, damping the excess

energy. Zeaxanthin accumulation puts the PSII antenna more on alert, ready to collapse into

the photoprotective state with high light exposure, which in turn lowers the PSII efficiency and

slows down the photosynthetic rates. However, the safety seems to be of a paramount impor-

tance for the photosynthetic membrane and the productivity is being firmly traded for it.

9.4.2.3 The Role of PsbS Protein in qE

Apart from the light harvesting complexes, a 22 kDa protein with four very hydrophobic

transmembrane helixes, called PsbS, was discovered to have a dramatic effect on qE. The

group of Niyogi isolated the mutants lacking this protein and found that they possessed

no rapidly-forming or relaxing NPQ: qE. The protein does not bind pigments and there-

fore is not likely to play a direct role in the photoprotective process. It was established

that PsbS possesses the two lumen-exposed glutamate residues which are essential in sens-

ing luminal protons. When the two glutamate residues on PsbS were mutated in

Arabidopsis plants ability to form qE was lost. Hence it was proposed that the protein

plays a role of a transducer of the trigger’s (ΔpH) action upon the antenna. This is a likely

scenario since PsbS was not found to be associated with the PSII supercomplex but rather

freely redistributed in the photosynthetic membrane. PsbS enhances qE even in the

absence of zeaxanthin and unlike the latter accelerates NPQ recovery. Hence, the action

of this protein is rather of a catalytic character: enhancing the dynamic range of NPQ,

basically, a qE-maker . Investigation of the structure of the PSII membranes by the freeze-

fracture microscopy combined with FRAP microscopy and detergent solubilization tech-

niques revealed a few specific effects of PsbS. Several striking features have been

discovered as a result of these studies. Firstly, PsbS affected the rigidity of the grana

membrane, its resistance to detergent solubilization and could accelerate the grana stacking

process induced by magnesium cations. Secondly, the absence of PsbS greatly enhanced

the occurrence of ordered semicrystalline arrays (see Figures 3.1 g, 7.1, 7.9) of PSII com-

plexes. Figure 9.17 (a) shows nearly two times enhancement of the occurrence of the

ordered PSII arrays in plants lacking PsbS. In the plants overexpressing the protein these

arrays were totally absent. Finally, PsbS decreased protein mobility in the grana

membranes as was first detected by Mullineaux and coworkers (Goral et al ., 2011) using

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the FRAP microscopy. Figure 9.17 (b) shows that the percentage of mobile protein in the

membrane in the dark for the mutant lacking PsbS was decreased, consistent with

the occurrence of semicrystalline arrays of PSII that are likely to enhance the membrane

rigidity. FPAP experiments also revealed that the onset of NPQ was followed by a strong

decrease in the protein mobility. The difference in this mobility between dark-adapted

and illuminated membranes with NPQ was the highest in plants overexpressing PsbS and

the lowest in those lacking the protein (Figure 9.17 ). These observations indicated an

existence of a structural change in the membrane that leads to the establishment of the

Figure 9.17 (a) Percentage of the membrane area occupied by the ordered PSII arrays in the grana, detected by freeze-fracture electron microscopy in the wild-type and PsbS-less (npq4) Arabidopsis plants. (b) Percentage of the mobile chlorophyll-protein fraction in the grana membranes of dark-adapted (D) and pre-illuminated to form NPQ (L) intact Arabidopsis chloroplasts. Courtesy of Tomasz Goral and Conrad Mullineaux.

(a)

(b)

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photoprotective state and a crucial role of PsbS in enabling this change as if the protein

acted as a ‘lubricator’ allowing PSII complexes to rearrange. It is, however, still not clear

why PsbS exerts this effect upon the protein mobility and whether its involvement in the

proposed structural change in the photosynthetic membrane is more specific than an

entropy-driven diffusion process.

9.4.2.4 The Role of LHCII Antenna Dynamics in qE: The Model of qE-Related Structural Changes within the Photosynthetic Membrane

The presence of a strong quenching in LHCII complexes upon their aggregation triggered

by low detergent concentration and low pH, modulation of this quenching and the aggrega-

tion process by the xanthophyll cycle carotenoids and sensitivity to activators (tertiary

amines, magnesium) and inhibitors (antimycin, cross-linkers) of qE suggested that its

mechanism involves aggregation of LHCII in vivo . In addition, qE was found to be accom-

panied by the changes in absorption, resonance Raman and fluorescence spectra that occur

during LHCII aggregation. Horton and Ruban ( 1992 ) proposed the LHCII aggregation

hypothesis for the control of light harvesting in the photosynthetic membrane of chloroplasts.

The ‘LHCII aggregation model’ shown in Figure 9.18 consists of the four different states

LHCII

Deepoxydation

Pro

tona

tion

Hypothesis

IV

II

III

heat

heat

heat

heat

I

Violaxanthin

Zeaxanthin

Figure 9.18 The LHCII aggregation model for NPQ. According to the Horton and Ruban ( 1992 ) model there are four different structural/functional states in the LHCII antenna, I, II, III and IV. I corresponds to dark-adapted, violaxanthin-containing unquenched state. Illumination causes violaxanthin de-epoxidation and protonation of LHCII, both driving the system into the deeply quenched state IV by promoting LHCII aggregation. Violaxanthin inhibits LHCII aggregation. If zeaxanthin is not formed LHCII will be only partially aggregated and quenched (III). After qE relaxation the antenna will still contain zeaxanthin and therefore remains partially aggregated and quenched (II), since it takes much longer for the epoxidation of zeaxanthin back into violaxanthin than for the relaxation of Δ pH. All four states therefore have different degrees of heat dissipation proportional to the degree of aggregation.

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of the LHCII antenna. The state I corresponds to a dark-adapted violaxanthin- containing

unquenched antenna. Illumination causes violaxanthin de-epoxidation and protonation of

the LHCII protein(s), both driving the system into the deeply quenched state IV by promot-

ing LHCII aggregation. Violaxanthin inhibits LHCII aggregation. If zeaxanthin is not formed

LHCII will be only partially aggregated and quenched (III). After qE relaxation the antenna

will still contain zeaxanthin and therefore remains partially aggregated and quenched (II),

since it takes much longer for the epoxidation of zeaxanthin back into violaxanthin than for

the relaxation of Δ pH. All four states therefore have different degrees of heat dissipation

roughly proportional to the degree of aggregation. Interestingly, the well-known small

absorption change at 535 nm associated with qE was found to originate from a red-shifted

zeaxanthin and violaxanthin. This shift takes place when the xanthophylls form J-type or

head-to-tail aggregates and can only take place during the promotion of interactions

between LHCII units: an aggregation process. Therefore the measurement of absorption

around 535 nm represents an only alternative to fluorescence method for monitoring the

establishment of the photoprotective state in the grana membrane.

The aggregation model of qE was not only consistent with many physiological, spectro-

scopic and biochemical observations but also explained the origins of zeaxanthin-dependent

qI components, the kinetic behaviour of qE, and the role of zeaxanthin as an allosteric

modulator of qE. The control of the sensitivity of qE to Δ pH by the xanthophyll cycle is a

process of great significance since in high light it allows qE formation at subsaturating levels

of Δ pH, which simultaneously allow high electron transfer rates, while in low light qE

is switched-off at levels of Δ pH which are still sufficient for ATP synthesis. Indirect estimates

of Δ pH in vivo suggest that the steady state levels of Δ pH are relatively low, explaining the

low levels of qE observed in the absence of zeaxanthin in vivo . Another important aspect of

the LHCII aggregation model is that it remains the only model that explains large variations

in NPQ levels observed among various plants and algae, since the fluorescence of LHCII

during the aggregation process, can be quenched up to 20 times. Therefore, the LHCII antenna

was proposed to possess an inherent property for the control of excitation energy density in

the photosynthetic membrane and for the fine regulation of qE sensitivity to the Δ pH.

The phenomenon of LHCII antenna aggregation in the photosynthetic membrane goes

beyond the qE mechanism. Formation of large aggregates of LHCII has been documented

in overwintering evergreen plants as a result of their long-term adaptation (acclimation) to

cold and high light. The process was followed by the appearance of a long wavelength

fluorescence characteristic of aggregated LHCII around 700 nm registered at 77 K (see

Chapter 8). LHCII aggregation has also been observed in plants grown under a CO 2 starva-

tion regime and with delayed senescence. The phenomenon of antenna protein aggregation

in the photosynthetic membrane seems to be of a universal regulatory significance and its

primary studies laid the foundation for various structural and spectroscopic studies deter-

mined to underpin the dynamics of the photosynthetic membrane landscape and its role in

the regulation of the light phase of photosynthesis.

Although the LHCII aggregation model has been supported by various experiments

discussed above it is only recently that the process of structural reorganization of PSII

Rupan_c09.indd 226 8/29/2012 5:35:28 PM


antenna has been visualized using electron microscopy approaches. It has been known

for a long time that the organization and lateral redistribution of photosynthetic com-

plexes in the thylakoid membrane depend upon cations. Magnesium is particularly cru-

cial for stabilization of grana stacking and the lateral segregation of PSI from PSII, as

well as the assembly of PSII–LHCII supercomplexes. Upon illumination formation of

Δ pH leads to neutralization of point charges on the lumen-exposed surface of the thyla-

koid membrane, resulting in displacement of bound magnesium cations and their diffu-

sion into the stromal space. Barber ( 1982 ) suggested that the alteration in charge

distribution brought about by Δ pH formation could cause alteration in the lateral interac-

tions and aggregation state of thylakoid membrane proteins. Holzwarth’s group

(Miloslavina, 2008) provided indirect spectroscopic evidence suggesting that upon for-

mation of qE part of the major LHCII undergoes separation from the PSII supercomplex

and aggregation. Bassi (Bassi et al ., 2009) obtained biochemical evidence suggesting

that the dissociation of a part of the PSII–LHCII supercomplex containing LHCII, CP24

and CP29 complexes occurred under NPQ conditions. Bassi’s group also found struc-

tural evidence using biochemistry and negative stain electron microscopy of isolated

thylakoid membranes that the distance between PSII core complexes decreased under

NPQ conditions in detergent solubilized grana membranes, implying that the onset of the

photoprotection involved a reorganization of the PSII antenna. Ruban ’s group obtained

ultimate structural evidence based upon freeze-fracture electron microscopy of intact

chloroplasts displaying clustering of PSII core units as well as LHCII antenna aggrega-

tion upon qE formation (Johnson et al ., 2011 ). Figure 9.19 shows the LHCII patterning

in the photosynthetic membrane based upon the analysis of the freeze-fracture electron

microscopy image using the image-processing software. The onset of the proton gradient

and violaxanthin de-expoxidation caused gradual change in LHCII coordinates so that

the particles formed progressively enlarging clusters: evidence of aggregation. The men-

tioned structural findings provided direct support of the hypothesis proposing LHCII

aggregation as a process underlying NPQ, and that Δ pH and de-epoxydation of violaxan-

thin to zeaxanthin cooperatively drives it. Moreover, the data shown in Figure 9.19 sup-

port the idea that qE and zeaxanthin-dependent qI have a common nature, LHCII

aggregation. Crucially, the structural alterations observed by Ruban’s group (Johnson et al ., 2011 ) induced by illumination occurred on a timescale consistent with the formation

and relaxation of qE. These data therefore provide the first direct link between the struc-

tural changes in PSII antenna and qE in intact, unsolubilized thylakoid membranes. The

observed clustering of LHCII complexes in the membrane is consistent with the FRAP

microscopy that revealed significant decrease in the protein mobility as shown in

Figure 9.17 . Indeed, clustering/aggregation of proteins cause formation of large areas

where protein mobility is almost absent as it is in the case of formation of ordered sem-

icrystalline arrays of PSII (see Figure 9.17 ).

The remarkable consistency between biochemical, structural and fluorescence micros-

copy experiments enabled Ruban and co-workers (Johnson et al ., 2011) to produce a

model presented in Figure 9.20 that summarizes all mentioned experiments regarding

the change in PSII–LHCII macro-organization underlying qE. In the qE state part of the

Rupan_c09.indd 227 8/29/2012 5:35:28 PM


PSII–LHCII supercomplex containing more loosely-bound trimers (L and M) and minor

antenna complexes CP24 and CP29 is dissociated, a phenomenon that relies upon the

presence of PsbS. This reorganization leads to the aggregation and partial special segre-

gation of LHCII from PSII. Unfortunately the resolution of the freeze-fracture work

precludes specifically assign the exact redistribution of each mentioned LHCII complex

or indeed PsbS to be known with certainty. This will remain a subject for the future

structural research using novel microscopy approaches. Zeaxanthin’s role in the qE

model is to promote the LHCII aggregation, as followed from the microscopy results

shown on Figure 9.19 . The higher aggregation state, the harder would be to return PSII

antenna system back to the light harvesting mode, hence the deceleration effect of zeax-

anthin upon the NPQ recovery kinetics. On the contrary, according to the model, PsbS

protein as an enhancer of fluidity should promote both, the formation and recovery of the

structural change underlying qE thus explaining its different from zeaxanthin effect upon

the process.

Experiment

VioLHCII

I

Zea

II

Vio + ΔpH

III

Zea+ΔpH

IV

Figure 9.19 Patterning of LHCII particles in the photosynthetic membrane determined by freeze-fracture electron microscopy (each LHCII trimer was fitted with a 50 nm 2 circle using image recognition software and their positions are presented in the figure), the four states of organization were observed in dark-adapted chloroplasts (Dark Vio, analogous to state I in the LHCII aggregation model), chloroplasts frozen immediately after 5 min illumination at 350 μmol photons m −2 s −1 in the absence of zeaxanthin (Light Vio, analogous to state III), chloroplasts frozen immediately after 5 min illumination at 350 μmol photons m −2 s −1 in the presence of zeaxanthin (Light Zea, analogous to state IV) and chloroplasts frozen following 5 min illumination at 350 μmol photons m −2 s −1 in the presence of zeaxanthin and a further 5 min dark adaptation (Dark Zea, analogous to state II). Reprinted from Ruban et al . © 2012, with permission from Elsevier.

Rupan_c09.indd 228 8/29/2012 5:35:28 PM


9.4.2.5 The Molecular Basis of qE Regulation

The changes in the lateral LHCII distribution were found to be accompanied by significant

conformational alterations, the thylakoid membrane became thinner. Figure 9.21 (a) shows

thin section negative stain electron microscopy of stacks of chloroplast grana membranes

where PSII is localized. The four micrographs correspond to the four states in the aggrega-

tion model of qE. Proton gradient as well as zeaxanthin will cause a significant decrease in

the membrane thickness as well as the space between stacked thylakoids. These observa-

tions are consistent with the model proposed by Packer (Murakami and Packer, 1970 ) of

the photosynthetic membrane response to the establishment of ΔpH. Figure 9.21 (b) illus-

trates this model. The flux of protons into lumen causes the countercurrent of magnesium

cations into the stromal compartment to balance the charge brought by protons. Membrane

components undergo dehydration and conformational changes in response to the proton

flux. The membrane loses water and becomes more hydrophobic and thinner. These

changes were found to be reversible and remarkably followed not ΔpH but rather NPQ

kinetics. This observation suggested a strong relationship between the changes in mem-

brane geometry and NPQ. Since LHCII is the major component of the grana membrane and

De-epoxidation

Dark / low light Excess light

Δ pH

PSII minor antenna

LHCII trimer

PSII core

Zeaxanthin

Violaxanthin

LHCII aggregate(quenched)

Figure 9.20 Scheme depicting the current knowledge regarding the structural reorganization of the PSII–LHCII macrostructure occurring in the NPQ state. Dissociation of the part of light harvesting antenna from PSII supercomplex leads to the reorganization and aggregation of LHCII complexes. Reproduced with permission from Johnson et al . © 2011 American Society of Plant Biologists. (See Plate 9.20 in colour plate section.)

Rupan_c09.indd 229 8/29/2012 5:35:28 PM


undergoes significant later rearrangements as shown earlier, the observed changes in the

membrane shown in Figure 9.21 are likely to involve enhancement in the antenna hydro-

phobicity. The synthesis of a most hydrophobic xanthophyll, zeaxanthin, resulting from

ΔpH formation further promotes this change. In addition, the LHCII aggregation process

itself enhances the average hydrophobicity of the protein.

In addition to the enhanced hydrophobicity of the grana membrane by protons and zeax-

anthin the role of PsbS as a cofactor of qE was further confirmed by the experiments that

used diaminodurene (DAD) and phenazine metasulfate (PMS) to enhance Δ pH across the

membrane in plants lacking this protein. Remarkably, these agents induced the rapidly-

forming and relaxing NPQ in the npq4 mutant. The pK of qE in the absence of PsbS (but

with zeaxanthin) was ~4.5 in comparison to 6.5 in the wild-type. It became clear that PsbS

simply is an efficient enhancer of antenna sensitivity to protons. The fact that qE can be

(a)

I

III IV

II

Figure 9.21 (a) The grana membrane ultrastructure studied by the thin section negative staining electron microscopy corresponding to the four states of the NPQ model shown in Figure 9.18 : I: dark + violaxanthin; II: dark + zeaxanthin; III: light + violaxanthin; IV: light + zeaxanthin intact spinach chloroplasts. Chloroplasts were fixed by glutaraldehyde and embedded in resin. Ultrathin-sections (~70 nm thickness) were cut and stained with uranyl acetate.

Rupan_c09.indd 230 8/29/2012 5:35:29 PM


obtained without PsbS suggests that the quenching process itself is independent of this

protein. Altered PSII–LHCII organization present in plants lacking PsbS (discovered in the

studies described in the previous paragraph) somehow shifts the Δ pH versus qE titration

curve to a lower pK, increasing the cooperativity of the process. Thus in the absence of

PsbS a larger Δ pH driving force (i.e. a more acidic lumen) is required to trigger the

reorganization of the PSII–LHCII macro-structure associated with qE (as discussed in the

previous paragraph). qE cannot be observed in npq4 plants simply because of the relatively

low levels of Δ pH that occur in natural conditions (pK of ~5.5–5.8).

But what is the mechanism of the effect of xanthophylls’ hydrophobicity and PsbS upon

the affinity of the LHCII structure for protons? The answer lies in the fact that apparent

pKa of amino acids strongly depends upon their environment. Hydrogen bonding, steric

Figure 9.21 (cont’d) (b) The model of the ∆pH effect on the photosynthetic membrane geometry and properties. Adapted from Murakami and Packer.© 1970 American Society of Plant Biologists.

(b)

Conformationalchanges

H2O

RH

Dehydration

130 – 135 Å

100 – 110 Å

Mg2+

H+H+

Outside(stroma)

Inside(lumen)

Increasedhydrophobicity

Decreasedthickness

Rupan_c09.indd 231 8/29/2012 5:35:32 PM


hindrance and the dielectric constant of the environment all affect the pKa of amino acids.

For example, the pKa of the carboxyl group on aspartate can be as low as 2.4, in water

environment due to hydrogen bonding, while in hydrophobic environment the pKa can be

as high as 6.4. The shift of qE sensitivity to Δ pH in the presence of hydrophobic xantho-

phylls such as zeaxanthin and hydrophobic proteins such as PsbS can be explained in this

way. The proximity of numerous acidic amino acid residues on the lumenal side of LHCII

(and CP29, see Chapter 6) to the xanthophyll binding domains provides a possible explana-

tion of the ability of xanthophylls to influence their pKa. Indeed, zeaxanthin binding shifts

the isoelectric point of CP26 to higher values. The careful regulation of the qE versus Δ pH

titration curve by xanthophyll de-epoxidation and PsbS is essential to maximize the

efficiency of photosynthesis (Figure 9.22 ). Since, the experimentally determined pKa of

the lumenal side of the thylakoid membrane is as low as 4.1, factors such as PsbS and

zeaxanthin which raise the pKa will allow efficient qE at lower values of Δ pH than

would otherwise be required but impossible to attain in vivo . The scheme presented in

Figure 9.23 explains the role of zeaxanthin and PsbS in qE by regulation of the sensitiv-

ity of the LHCII system to protons. Without zeaxanthin and PsbS the pK for LHCII

protonation is very low (~4.0) and therefore the complex(es) remain(s) unprotonated and

qE is absent. The isoelectric point of PsbS is much higher than that of LHCII at ~6.0 thus

the protein responds to even low levels of Δ pH. Protonation of PsbS is known to promote

Z V

Physiological lumen pH~5.5–5.8

Z+PsbS V+PsbS

LHC

II reorganisation

Lumen pH

80.8

0.6

0.4

0.2

qE

0.00 1 2

Δ pH

3 4 5

7 6 5 4 3

Figure 9.22 ∆pH titrations of qE in intact Arabidopsis chloroplasts containing veaxanthin and PsbS (Z + Psbs), violaxanrthin and PsbS (V + PsbS), lacking PsbS but containing zeaxanthin (Z) and lacking PsbS and containing violaxanthin (V). The average lumen pH in vivo is marked by a dashed arrow.

Rupan_c09.indd 232 8/29/2012 5:35:32 PM


the observed reorganization of the PSII–LHCII macrostructure in excess light leading to

LHCII aggregation and this in turn may increase the pK for LHCII protonation by

making the complex more hydrophobic when it aggregates.

The pK for the activation of the violaxanthin de-epoxidase is ~6.0 and the formation of

zeaxanthin then further promotes LHCII aggregation such that the pK is further shifted up

to ~5.7–6.2 allowing protonation at moderate levels of Δ pH thus amplifying qE. Hence,

hydrophobicity and aggregation (reorganization) of the PSII antenna are mutually enhancing

processes favouring the establishment of its quenching conformation. As was demonstrated

in Chapter 8, aggregation of LHCII complexes is not obligatory requirement for the

induction of quenching in vitro .

Hence, the nonphotochemical quenching originates from the individual complexes by

formation of the quenching pigments within the LHCII structure. This means that the

observed in vivo cooperative nature of qE in the absence of zeaxanthin and/or PsbS arises

from the tendency of the protonated LHCII conformation to aggregate. As LHCII begins to

aggregate so the hydrophobicity of the structure and thus affinity for protons increases, thus

proton binding is cooperative. By promoting aggregation zeaxanthin and PsbS thus reduce

the cooperativity of the proton binding equilibrium.

Tuning the photoprotective response is ecologically important, since different light envi-

ronments require different light-tracking strategies within a light harvesting system. LHCII

with more hydrophilic xanthophylls and faster NPQ recovery would be more desirable at

very frequent but low amplitude fluctuations of light intensity. On the contrary, LHCII

carrying hydrophobic xanthophylls with the tendency for NPQ with slow recovery would

LHCII

LHCII

H+

qE

Hydrophobicity Aggregation

PsbSVDE

4.0 6.06.0

>6.0

Figure 9.23 Scheme explaining the effect of PsbS and xanthophyll cycle on the pKa of qE-active residues in LHCII. The pKa of LHCII is ~4.0, too low for qE activation by physiological lumen pH values 5.5–5.8, however PsbS and violaxanthin de-epoxidase have a pKa of ~6.0 and thus bind protons, together they trigger the aggregation of LHCII increasing the hydrophobicity of the environment of the qE-active residues and shifting the pKa to ~6.0 thus activating qE at physiological lumen pH values. Reprinted from Ruban et al . © 2012, with permission from Elsevier.

Rupan_c09.indd 233 8/29/2012 5:35:33 PM


be better suited to slower but stronger fluctuations in light environment. The xanthophyll

cycle fulfils the LHCII tuning role in vivo .

9.4.2.6 Formation and Identity of qE Quencher(s)

We have so far discussed the site and change in the PSII membranes associated with the

establishment of Δ pH and qE state. The change is apparently important to study in order to

understand, first of all, the principles of qE regulation as a basis for the light management

by plants on the molecular level. It became apparent that aggregation of LHCII antenna in

the membrane is an important process, largely thermodynamically driven, that is essential

for maintaining the fine balance between antenna efficiency and photoprotection. This

regulation ensures optimal electron transfer regime in PSII and hence productivity of the

light phase of photosynthesis. Another important aspect of nonphotochemical quenching is

what are the events in the individual protonated LHCII antenna complexes that lead to

quencher formation on the atomic scale? In other words, the question of what is the qE

quencher and how it is ‘born’ in the antenna as a result of protonation followed by the

antenna rearrangements and aggregation remains to be discussed.

Unfortunately, the full atomic picture of changes in protein that create the quencher is

not available yet. The structure of a quenching state of LHCII exists (see Chapter 8),

however, no structure is available of LHCII in highly fluorescent, unquenched state or, at

least, the one with the bound zeaxanthin instead of violaxanthin. There are a few lines of

evidence that revealed some structural alterations within individual LHCII complexes

associated with the onset of NPQ. First, is that cross-linkers were found to inhibit the

process in vitro as well as in vivo (see previously and also Chapter 8). These reagents bind

to various aminoacids of protein, interlinking them within the structure that causes liter-

ally ‘freezing’ it in a particular structural state. Second, the high hydrostatic pressure was

discovered to promote the quenching in isolated LHCII that is likely due to an effect on

the protein. Thirdly, thermodynamic studies of the quenching formation in LHCII in vitro

and qE yielded energy parameters (activation energy, etc., see Chapter 8) indicative of

protein dynamics on the energy scale that corresponds to a several hydrogen bonds.

Fourthly, resonance Raman spectroscopy revealed a specific conformational change in

neoxanthin that reflects some structural alterations within the protein during the onset of

quenching in vitro and in vivo .

The quencher identity was researched by various spectroscopies of isolated LHCII

dynamics. Only a few specific features associated with both, quenching in isolated LHCII

and qE have been revealed. Both types of quenching were characterized by the appearance

of the red-shifted Chl a band(s) absorbing above 680 nm and a decline in absorption of a

several bands belonging to xanthophylls. Time-resolved fluorescence spectroscopy revealed

that qE and quenched state of LHCII are both characterized by red-shifted room tempera-

ture fluorescence components. Based on the spectral changes described it was proposed that

the qE quencher originates via the promotion of pigment–pigment interactions within the

LHCII complex. Homo- or heterodimers of chlorophyll have been proposed to be involved

in the quenching. Indeed, chlorophyll associates, permanent or transient, were known to

Rupan_c09.indd 234 8/29/2012 5:35:33 PM


possess very low fluorescence yield and similar spectroscopic features to those observed in

aggregates of LHCII (see Chapter 5). The concentration-type of quenching that the LHCII

design was evolved to conquer emerged at NPQ state, when the protein structure was altered

by protonation. Figure 8.2 of Chapter 8 shows the configuration of Chl a610–612 in the

terminal emitter locus – likely candidates for the proposed quenching Chl–Chl associations.

There are a few other chlorophyll dimers that can be found in the LHCII structure that may

well represent the qE quencher. The mechanism of quenching in chlorophyll dimers can be

of a charge transfer character since the quenching in vivo and in vitro is associated with the

red-shifted fluorescence with a strong temperature dependency discussed above and in

Chapter 8. At the moment, the theoretical work is being carried out to find out if chlorophyll

associates can be accounted for the quenching in LHCII.

Apart from chlorophylls, the excitation energy can be taken and quickly dissipated

from the terminal emitter by xanthophylls. The physical mechanism of xanthophyll

action as excitation quenchers in LHCII antenna was proposed to involve S 1 (2Ag)

state. The S 1 possesses an extremely short lifetime (~10 ps) likely due to the crossing

of its potential energy surface with that of the ground state (at a so called conical inter-

section). The short lifetime and close proximity of the S1 state energy to that of the

lowest excited state of chlorophyll in the Qy band arguably makes xanthophylls ‘natu-

ral born quenchers’. However, the S 1 state has the same spatial symmetry as the ground

(S 0 or 1Ag) and, in accordance with the selection rules for electronic transitions, is

dipole-forbidden resulting in the absence of the absorption. This complicates the

energy transfer relations with chlorophyll and remains a challenging subject for exper-

imental and theoretical studies. However, excitonic coupling of xanthophylls to chlo-

rophylls have been detected that was correlated with qE quenching. Excitonic coupling

is realized between two pigments when the exciton transfer integral coupling the two

molecules (a measure of the energy interaction between the electronic transitions of

the two molecules) is much greater than the dephasing energy (a measure of the inter-

action between a molecule and its environment). Two pigment molecules are said to be

excitonically coupled if excitation energy can be coherently transferred between them.

The molecular dimer then behaves as a single quantum mechanical entity and the exci-

tation is delocalized across both molecules. In principle, such excitonic interactions

could result in low-lying states that possess more carotenoid than chlorophyll character

showing enhanced coupling to the ground state, thus making them efficient quenchers.

It is possible however that a distortion of the xanthophyll brought about during quench-

ing could allow direct excitation of S 1 by removing the symmetry restriction that makes

the state forbidden.

Currently there are two models proposed involvement of xanthophylls in NPQ. The first

model is based upon the structural dynamics of the minor LHCII complex, CP29. It

suggests that zeaxanthin converted from violaxanthin at lutein 2 site of CP29 complex

forms a quenching association with the two chlorophylls, a 603 and b 609 (Figure 9.24 a).

Similar scenario was proposed to take place in the other minor antenna complexes, CP24

and CP26. When two molecules are excitonically coupled the lowest lying excited states

mix resulting in two delocalized excitonic states and additionally two charge transfer states

Rupan_c09.indd 235 8/29/2012 5:35:33 PM


which correspond to the hole (the positive part of the exciton) and the electron being local-

ized to individual molecules within the dimer. For a homodimer, that is, one for which the

individual molecular excited states have very similar excitation energies these charge trans-

fer states lie above the excitonic states. Conversely a heterodimer composed of molecules

with very different excitation energies the charge transfer states generally lie below the

excitonic states. Under certain circumstances it is energetically favourable for the charge

transfer state to dissociate via charge separation into an anion and a cation. The hole and

electron that make up the exciton are no longer bound together and the exciton is destroyed,

charge recombination between the anion and cation follows and the energy is dissipated as

heat. A xanthophyll radical cation signal with a rise time of ~11 ps, attributed to zeaxanthin,

was detected in the qE state upon chlorophyll excitation by Fleming and coworkers (Holt

(a)

CP29–V + H+= (CP29–Z – H+)

Z

b609

a603

LHCll–V/Z + H+= (LHCll–V/Z–H+)

a610

a611

a612Lut1

Neo

(b)

Figure 9.24 Atomic structure level models of possible NPQ sites. (a) The minor LHCII quenching model propose that protonation of the PSII minor antenna complexes leads to a conformational change leading to formation of a xanthophyll–chlorophyll quenching interaction between zeaxanthin bound at the lutein 621 site and chlorophylls a603 and b609. (b) The major LHCII quenching model propose that protonation of LHCII leads to a conformational change causing a quenching interaction between the lutein bound at the lutein 620 site and chlorophylls of the terminal emitter, a610–a612. Neoxanthin distortion is a spectroscopic signature of this conformational change.

Rupan_c09.indd 236 8/29/2012 5:35:33 PM


et al ., 2005 ) using transient absorption spectroscopy (for the method details see Chapter 8).

The same zeaxanthin cation signal was observed in isolated PSII minor antenna complexes

and was shown to be dependent in the case of CP29 on the presence of chlorophylls a603

and b609 in close proximity to the xanthophyll bound into lutein 621 site (Figure 9.24 a). It

was thus suggested that the charge transfer state forms by delocalization of the electron

across the two chlorophylls. Recently, lutein cations have also been detected in the PSII

minor antenna proteins and these have been suggested to play a role in quenching, particu-

larly under circumstances when zeaxanthin is absent.

The second model of xanthophyll involvement in qE is based upon the major LHCII (but

does not principally exclude the minor antenna) (Figure 9.24 b). The model proposes small

conformational alteration within the monomeric unit of the complex that causes twist in

the neoxanthin molecule (shown by the bent hollow arrow) and simultaneous (cooperative)

movement of lutein bound to L1 site (shown by three straight arrows). Since this lutein is

very closely located to the terminal emitter locus containing Chl a 610–612 it is proposed

that protonation that causes the change in the protein structure brings lutein close enough

to these pigments (almost at the van der Waals contact) to act as the terminal emitter chlo-

rophyll excited state quencher. Transient absorption studies on LHCII aggregates performed

by van Grondelle’s group (Ruban et al ., 2007 ) also revealed the transient population of a

xanthophyll S 1 state upon chlorophyll excitation (see Chapter 8). The position of the S

0 –S

2

bleach in the TA kinetics indicated that the species involved was lutein 620 (Lutein 1)

rather than zeaxanthin which was absent from the prepared complexes. According to the

model shown in Figure 9.24 (b) the conformational switch in LHCII structure opens a

channel for energy transfer from chlorophyll to the lutein S 1 state. Such a mechanism

would invoke incoherent coupling between the states for which the coupling between

molecules is much weaker than the coupling of each molecule to its local environment (the

opposite limit to excitonic interactions). Transfer of energy between them is thus said to

occur ‘incoherently’, hopping from one to another while at any time being localized on a

single molecule, the short lifetime of the xanthophyll S1 state in this case making it an

efficient quencher. Förster ( 1948 ) classically described incoherent energy transfer between

dipole-allowed states, via interactions between the transition dipole moments of each

molecule. By this logic, Förster transfer to the forbidden xanthophyll S 1 state cannot occur.

Generalized Förster theory, however, which takes into account the size of the molecule

does permit transfer to and from forbidden states. Alternatively there is the exchange inter-

action-mediated Dexter mechanism that describes the incoherent transfer of energy

between two molecules whose electronic orbitals closely overlap, allowing electron

exchange (see Chapter 5).

Discussions about the exact photophysical origins of the quencher thus remain the sub-

ject of great debate in the NPQ field. New approaches in quantum mechanical modelling of

the excited states of carotenoids and chlorophylls should provide new insights in the future

into the complex interactions of these molecules. While, new experimental techniques such

as femtosecond stimulated Raman spectroscopy may reveal new details of the fates of

excitation energy in quenched systems.

Rupan_c09.indd 237 8/29/2012 5:35:34 PM


9.4.3 Future of qE Research

Four key elements of qE: trigger , site , change and quencher were defined and discussed

here in their connection with dynamics and regulation of this process. It must be said that

there exist several important points in qE research to be addressed in order to build a

complete picture of the mechanism of this outstanding, intriguing, complex and long

investigated process. Among them are the following: is there a specific complex respon-

sible for qE or can the process take place in all LHCII complexes, major and minor?

Could genetic manipulation approaches help to solve this question? Is there only one qE

quencher or several? What is (are) the precise photophysical mechanism(s) of quench-

ing? How effective is the qE process in the photoprotection of photosystem II and for the

crops in general? Is the work on mutations in antenna pigments and proteins feasible for

the use in agriculture? In our view, the answers to these questions may not be as clear cut

as one desires. For instance, while qE appears to be a single process that is regulated in a

rather complex way, one cannot exclude that the quenching process itself may originate

from a nonspecific environmental effect that is felt by all pigments. The fact that

the quenching can exist in both, minor and major antenna complexes, and the evidence

implicating different sets of pigments in quenching is consistent with such a view.

The future development of new mechanistic approaches and further integration of the

disciplines of biology, chemistry and physics could shed light upon these and other

important questions related to the one of the most remarkable mechanisms of photo-

protection and regulation of the light phase of photosynthesis.

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Ruban , A.V. , Young , A. , Pascal , A. and Horton , P. ( 1994 ) The effects of illumination on the xantho-phyll composition of the photosystem II light harvesting complexes of Spinach thylakoid membranes . Plant Physiol. , 104 , 227 – 234 .

Tikkanen , M. and Aro , E.-M. ( 2012 ) Thylakoid protein phosphorylation in dynamic regulation of photosystem II in higher plants . Biochim. Biophys. Acta , 1817 , 232 – 238 .

Siffel , P. and Vacha , F. ( 1998 ) Aggregation of the light-harvesting complex in intact leaves of tobacco plants stressed by CO2 deficit , Photochem. Photobiol. , 67 , 304 – 311 .

Weis , E. and Berry J.E. ( 1987 ) Quantum efficiency of Photosystem-II in relation to energy-dependent quenching of chlorophyll fluorescence . Biochim. Biophys. Acta , 894 , 198 – 208 .

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Numberless noisy weathercocks rattled and sang of mutation . from Evangerline: a tale of Acadie, Henry W. Longfellow

10.1 Science and Society

The relationship between science and society has always been uneasy. Academic science

first emerged in Europe under the wing of religious institutions promoted by their spiritual

and cultural desires. Natural science appeared later, when the society realized its sheer

potential to enrich and empower. However, the thirst of man for knowledge is a rather

ancient instinct if not evolutionary. One can argue that the ‘wonder’ reflex has been

developed to an advantage of survival, aiding exploration and proliferation of humans in

the natural environment and shaping the emerging ability for abstract thinking. The

reaching for knowledge is therefore an important inherent and objective attribute of human

life for the individual as well as societal. In a way, this attribute is as rightful and objective

as religion having an institutional status of the human nature. Society despised science and

scientists in times when it felt in danger of them and cheered and supported research

activities when it wanted to exploit their results in medicine, architecture, warfare,

agriculture and other spheres of its life. Society witnessed great power of science in

industrial revolution, militarization and cure of diseases. Fruits of science, therefore,

altered the face of human history and likely to alter the face of the whole evolution of our

Biosphere. The relationship between science and society is a crucial determinant of man-

kind’s fate. One of the major reasons why it became possible for our planet to sustain an

What is in it for Plant, Biosphere and Mankind?

10

Rupan_c10.indd 241 8/29/2012 5:34:52 PM


enormous population growth is science. The reason for a great technological progress is a

progress in natural sciences. The latter, in turn, relies upon the former and the willingness

of society to support certain research areas for its benefits. It feels as if the science is an

eternal flame, an inexhaustible carrier of energy and society is a manager of this energy as

well as its customer. The progress in the particle physics would not be the same if not

driven be the defence purposes of society. The Large Hadron Collider would not have been

built if there was no visible practical outcome expected from it. Science owes society for

the possibility to carry on research otherwise unthinkable. The modern science became a

part of various industries, collective, dependent, directed: more organized and civilized

than in the times of inquisition. The opportunities in research for many are great and the

challenges are unprecedented.

The major problems society wants natural sciences to address in modern days are

health , food , peace and climate . The sequence in which these four are listed reflects per-

haps the order of their importance for society. Naturally, medical sciences are champions

in fundraising. Food and defence research follows. The investment in understanding the

environmental changes seems to be less crucial; however, it may well turn out to be the

most important priority of all. The understanding of the impact of man on the environment

and its consequences is not straight-forward and full of controversies. Hence, the research

support is needed. The society acts, however, as a very busy manager, trying to solve

‘burning’ issues (health, etc.) leaving others for later, perhaps when they themselves

become ‘burning’. However, whilst the average health of mankind is such that its popula-

tion is on a steep rise, the global character of environmental changes has a chance to lead

to its dramatic decline and death of our civilization. Some argue that the observed global

warming is a process caused by man. A solid fundamental research is needed to predict

the rate and consequences of this process and, most importantly, avert its detrimental for

the all life on our planet effect. If man is responsible it is also our moral duty to safe the

richness and beauty of the world we live in. Hence, the promotion by society of basic

natural sciences could be of enormous help, provided society will allow that. In the sci-

ence of photosynthesis and, in particular, Sun energy capture, one could see a great power

that could be used to save the planet Earth’s Biosphere and teach the future generations to

live in harmony with it.

10.2 Energy Balance of Photosynthesis: A Wasteful Process?

Photosynthesis is by far the major process that supplies energy to Biosphere. Hence, it is

the major source for battling entropy and sustaining life on Earth (see the Chapter 1). Life

simply cannot exist without the input of energy and with it and adequate environmental

conditions it can be fairly autonomic, evolving for millions of years, learning ways to adopt

and proliferate in space. The events in the photosynthetic membrane relate only to the light

phase of the process (see Figure 3.7, Chapter 3) ending with the synthesis of ATP and

NADPH. The dark phase uses their energy to produce glucose. The whole process of pho-

tosynthesis is a remarkable cascade of energy transformation reactions that deals with a

Rupan_c10.indd 242 8/29/2012 5:34:52 PM

What is in it for Plant, Biosphere and Mankind? 243

range of types of matter: photons , electrons , protons , molecules (see Figure 1.1, Chapter 1).

Hence the energy losses are inevitable and could be large. It is important, however, to point

out that some of the losses are avoidable and some are not necessarily unwelcome. The

following paragraphs will analyse each type of loss, explaining their nature and potential

variability. The question of energy balance seems to be a major question related to the

importance of photosynthesis for the mankind and the Biosphere.

In order to assess the light energy utilization efficiency by nature it is of advantage to

show its whole path from the Sun to glucose , the final product of photosynthesis. The

efficiency of the photochemical process of CO 2 assimilation is limited due to the losses on

the way of transformation of the energy of redox-reactions in the photosynthetic membrane

in to the chemical energy stored in glucose. The losses are around 68% as highlighted by

the grey band on the light energy efficiency diagram depicted on the Figure 10.1 . These

losses can be assessed as following. A minimum eight photons of the energy matching the

absorption of PSII and PSI reaction centres (680 and 700 nm) are required to fix one mol-

ecule of CO 2 . To fix six molecules of CO

2 in order to build one molecule of glucose,

C 6 H

12 O

6 (see Equation 1.2, Chapter 1), it would take for photosynthesis 48 such photons:

24 with the energy of 176 kJ mol −1 (680 nm) and 24 with 182 kJ mol −1 (700 nm) totalling the

energy of 8592 kJ mol −1 . However, 1 Mol of glucose stores only 2801 kJ of energy. Therefore,

the efficiency of the photochemical synthesis of glucose in the process of photosynthesis is

about 32%. Hence the inevitable losses of energy storage in the process of photosynthesis

as we know it are about 68% (Figure 10.1 ). These losses cannot be reduced unless someone

could improve Nature itself and come up with a better photochemical system possessing

higher efficiency.

However, out of 100 000 TW of available solar energy, only about 100 TW are stored in

Biosphere by photosynthesis. This means that the efficiency of the process in nature is 0.1%,

that is to say, some 300 times lower than the calculated efficiency of the photochemical

synthesis of glucose. Why is that? Why photosynthesis is such a wasteful process in the

‘real life’? Why nature did not invent a better one? Contemplating on this point makes one

realize that life’s ‘ambitions’ to concur the space and adapt to various conditions could also

do with having better, more efficient ways of gaining energy from the environment. Instead,

life in the Biosphere became rather extremely diverse then economic, with complex rela-

tionships between its different forms. Interestingly, mankind often tended to trend this

diversity for efficiency and power, as well as the rate of development. This led first to the

huge economic boom within the recent 100 years resulting now in approaching energy and

environmental crises.

Let us consider some other factors that undermine so greatly the efficiency of photosyn-

thesis. Figure 10.1 represents the light energy losses in the photosynthetic membrane, plant

as well as in the environment (atmosphere). The arrowed horizontal lines highlight the

levels of energy remaining after a certain type of loss has occurred. The vertical arrows

show their approximate variation ranges. The line above the photosynthetic CO 2 assimila-

tion losses of ~68% marks a relatively low level of energy on the energy bar on the right

with 0.28 fraction of the unity (highlighted by the thick grey line). The latter corresponds

Rupan_c10.indd 243 8/29/2012 5:34:52 PM


to the level of light energy that enters the plant. This level itself is extremely variable and

is determined by the two types of factors that cause: (a) constant light losses due to the

absorption in the upper atmosphere and by gases and (b) variable light losses due to light

scattering in clouds and absorption by canopy. The (b) is variable because the transient

nature of the factors that cause them (see Chapter 9). Whilst the constant light losses in

atmosphere are about 20%, the variable losses could reach one or even two orders of

magnitude (dense canopy in the tropical forest, water absorption in aquatic photosynthesis,

etc.). The variable light losses greatly determine the evolution and diversity of not only

plants but all photosynthetic organisms, causing divergent and often independent

development of the photosynthetic light harvesting antennae as well as the mechanisms

that cope with the variable or even sudden high light exposure, like NPQ (Chapter 9).

Transient losses of light represent the environmental factor providing photosynthetic

organisms with physiological challenge that plants have to face. Clearly these losses should

inevitably affect the global energy storage in the Biosphere by various organisms having

variable share of 100 000 TW of light available from the Sun.

spectral availability

excitation energy delivery

electron/proton transportand CO2 assimilation

respiration processes,growth,etc.

constant lossestransient losses

clouds upper atmospheregasescanopy

~47%

1.00

0.70

0.37

0.28

0.090.05

~24%

~68%

~30%

~20%up to 99%

scattering in tissue/cell/chloroplast

Env

ironm

ent

Pla

nt

Str

oma

Ph

oto

syn

thet

ic m

emb

ran

e

Lig

ht

har

vest

ing

Figure 10.1 Light energy losses in the process of photosynthesis in Nature.

Rupan_c10.indd 244 8/29/2012 5:34:53 PM


Light energy losses in the plant itself (below the thick grey line) start from the losses due

to light scattering in the photosynthetic tissues, in cells and chloroplasts themselves until

light reaches the photosynthetic membrane. These losses are approximately about 30%.

Next loss of light energy comes from the fact that the photons of different energy (colour)

are differently absorbed by the photosynthetic pigments (see Chapter 5). This means that for

plants mainly blue and red photons are photosynthetically competent or active. Hence, the

name of the spectrum of light used in photosynthesis: PAR or Photosynthetically Active Radiation . Figure 10.2 shows the Sun radiation spectrum near the surface of Earth and a

spectrum of PAR that could be actually used by plant (dotted line). Calculations show that

the fraction of PAR is about 53%, hence the loss in the photosynthetic efficiency is about

47% of the light available after scattering in tissue, and so on. There is not much room for

variations in the fraction of PAR in higher plants, however, the parameter varies among dif-

ferent photosynthetic organisms simply because the differences in their pigment types,

hence the absorption spectra of their photosynthetic membranes. The loss of 47% of this

energy level lowers the efficiency to the level of 0.37 (0.7 × 47%). From this point light

enters the light harvesting antenna. As was previously explained, the photosynthetic antenna

is a great evolutionary achievement of nature, enabling the existence and survival of the

photosynthetic organisms in very low light environment. It is because of antenna the PAR is

almost a half of the sunlight spectrum, not just a small fraction of it, since the antenna broad-

ens the absorption cross-section of the photosynthetic reaction centres. An interesting exam-

ple of the spectral broadening of photosynthetic cross-section was discussed in the Chapter

8 (Figure 8.12). In plants lacking the major polypeptides of the LHCII complex, CP26 minor

antenna complexes formed trimers, mimicking the oligomeric state of the native LHCII. The

formation of trimers shifted the absorption of one of the lutein molecules (Lut 621) to the

red by 15 nm. This apparently small change increased the spectral cross-section of the newly

built PSII antenna. Hence, the described oligomerization process of antenna proteins is

Figure 10.2 Spectra of available energy from the Sun near the surface of Earth (solid line) and typically utilized in photosynthesis of higher plants (dashed line).

Rupan_c10.indd 245 8/29/2012 5:34:53 PM


essential for ensuring the interception of light of a broader spectral range. This contributes

little to the total cross-section of PSII reaction centre but globally, 5% of extra captured

energy would be ~5 TW of energy: the value corresponding to the combined US and EU

yearly energy demands. A small adjustment on the molecular nanoscale can easily have a

large impact on the scale of the entire Biosphere. Therefore, the knowledge of the funda-

mental principles of design and functioning of the photosynthetic membrane is crucial for

our understanding of the impact of plants and other photosynthetic organisms upon the life

on our planet.

The next loss of light energy occurs in the photosynthetic antenna after absorption of

PAR. This loss is due to the fact that although the antenna is tuned to have a broad spectral

cross-section, only energies that correspond to approximately 680 and 700 nm absorption

of PSII and PSI reaction centres can be utilized. Figure 5.18 in Chapter 5 clearly illustrates

this point and indicating Δ E as the energy loss incurred in a process of energy transfer in

the antenna first to the terminal emitter and then to the reaction centre . This loss in the

photosynthetic membrane is estimated to be around 24% (Figure 10.1 ) and is solely due to

the organization and types of photosynthetic pigments. The vertical arrow corresponding to

the variability of these losses points mainly downwards indicating that the 24% is a basic

minimum and can be significantly larger in certain cases. Chapter 9 described the phenom-

enon of saturation of energy trapping in PSII reaction centres due to the limited turnover

rate. It is well-known that such saturation can occur in many plants at relatively low light

intensities (~10% of the full sunlight), revealing the major cause of photosynthetic

inefficiency. The saturation light intensity can vary among plants by more than 20 times

and the variations in the electron transport rate via PSII can be as high as the one order of

magnitude. At the conditions when the absorbed energy by antenna cannot be used for

charge separation and electron transport it is eventually wasted as heat, that diminishes the

photosynthetic yield. The onset of NPQ in antenna helps to dispose of the unused energy

quicker, saving the photosynthetic membrane from the photodamage. The decrease in the

photosynthetic efficiency due to NPQ is transient, protective and therefore a positive event.

Nevertheless, NPQ and photoinhibition, both strongly diminish the average photosynthetic

efficiency.

Finally, additional energy losses occur during and after CO 2 assimilation stage of photo-

synthesis via loss of electrons into respiration processes and loss of glucose for growth,

transport and other energy requirements of plant life. These bring down the final figure of

light energy efficiency storage to well below 1%. As was shown earlier, this figure is rather

close to 0.1% globally.

Figure 10.1 clearly shows that by far the major losses of light energy in photosynthesis

occur in the process of light harvesting and charge separation: not all photons are being

suitable, some lost before absorption, others lost in the processes of transfer and trapping

and lots are absorbed but not used at all due to the saturation of electron transport. One can

argue that evolution made the antenna potentially very efficient, able to supply sufficient

energy to the photosynthetic machinery in deep shade, yet not capable of collecting of all

light photons at even moderate light intensities and forced to ‘waste’ energy via NPQ in

Rupan_c10.indd 246 8/29/2012 5:34:53 PM


order to avoid photodamage. This protective measure results from the electron transport

saturation. The latter seems to be inevitable, since the ‘slowing down’ and accumulation

into the chemical form (glucose) of the captured light is the goal of photosynthesis that

satisfies the metabolic requirements of life on our planet (see Chapter 1). Photosynthetic

antenna can therefore be both, extremely efficient and inefficient and such flexibility offers

a potential for adaptations as well as its artificial manipulation.

10.3 Crops and Light Harvesting

Crops are the result of artificial selection by the mankind. The agenda was different here

than that of the natural selection, since the latter was aimed at survival of plants whilst the

former at survival of humans. As a result, crops are generally weaker organisms, reliant

upon fertilizers and pesticides. However, crops are constantly being pushed to produce

more. This requires, above all, energy that comes from light. Does light harvesting have to

be different in crops from that of wild plants? The Figure 10.1 scheme suggests that the

process inefficiency is not a limiting factor, if anything; it needs to be flexibly made very

inefficient to protect the photosynthetic membrane against damage. But the protection,

NPQ, will inevitably lead to decrease in efficiency, but safe, rather than one that is very

expensive to restore (photoinhibition). Unless electron transfer and carbon assimilation

rates are made faster the light harvesting improvement is not of a concern. Nature evolved

the antenna in order to enable the photosynthetic organisms to survive in environments

with very low light. Therefore, the light capture is effective enough to satisfy the require-

ments of electron transport and can indeed saturate it at relatively low light intensities.

Some plants like the fastest-growing Phyllostachys pubescens simply make use of very

large canopy (leaf index area of 8–12) that enables it to harvest almost all available light

(Figure 10.3 ). However, plants vary in the electron transport and reaction centre turnover

rates as was mentioned in the previous paragraph. The causes and regulation processes

involved are not very well understood. The alterations in stoichiometry of some electron

transport caries are one of the factors that affect the light saturation curves. How, for

example, the RCII turnover relates to the plant productivity is also not a simple question.

In theory, they should correlate but the type of this relationship depends on various

environmental and metabolic factors. Interestingly, faster turnover would accelerate the

earlier onset of photoinhibition, since it seems that the latter depends upon the number of

turns rather than light intensity per se .

The dynamic nature of NPQ regulation seems to be an important aspect of light harvesting

in crops. Indeed, it is not enough to have just large NPQ, but it is absolutely vital to make

sure that it closely tracks the light intensity fluctuations. Even for many crops growing in

the open field the intermittent shading within their tightly grown three-dimensional canopy

makes the light exposure very much agile. To track the fluctuations NPQ must be of a large

amplitude as well as rapid formation and recovery rate. As was discussed in Chapter 9,

zeaxanthin amplifies qE, a quickly-reversible part of NPQ, making it faster to form but

slower to recover. On the other hand, PsbS protein accelerates greatly both, the formation

Rupan_c10.indd 247 8/29/2012 5:34:53 PM


and recovery of this process. Therefore, for crops enduring large and frequent (in a timescale

of seconds) fluctuations of light, high levels of zeaxanthin as well as PsbS protein would be of

benefit. In nature the levels of xanthophyll cycle carotenoids as well as PsbS protein can vary

by several times, suggesting that plants have an inherent potential to acclimate (long-term

adaptation) to the different light habitats by altering the composition of these key regulatory

molecules in the photosynthetic membrane. Hence, genetic manipulation of PsbS expression

as well as the genes of xanthophyll biosynthesis enzymes became a feasible strategy these days

in the pioneering work on the crop improvement programs worldwide.

An additional critical point of light harvesting regulation in crops is the efficiency of

NPQ in photoprotection. One has to assess how well plants are protected against

photoinhibition by quenching. The latter can be very fast to form and recover but not large

enough to offer effective protection. Or, the quenching can be too great, wasting a part of

excitation energy in the antenna that could have otherwise been used in photosynthesis. To

address the protective requirement in NPQ, the work on development of methodologies to

assess the process, establish long-term monitoring methodologies and manipulate it accord-

ingly is necessary. The strategies of light harvesting control for different types of crops are

individual and, for those growing in greenhouses are very different from those required for

growing outdoors. The main peculiarity of the greenhouse culture is the possibility to grow

under constant illumination. Since, as was discussed earlier in this chapter, even moderate

light intensity can saturate photosynthesis, one can potentially think of adjusting the growth

Figure 10.3 Phyllostachys pubescens , the efficient light harvester and one of the fastest growing plants. Kyoto Rakusai Bamboo Park.Courtesy of Kateryna Law. (See Plate 10.3 in colour plate section.)

Rupan_c10.indd 248 8/29/2012 5:34:54 PM


light intensity to the level that is just enough to saturate electron transport, avoiding strong

photoinhibition and on the other hand having controlled levels of NPQ, just necessary to

dissipate the excess light, if any. In the perfect scenario, it would be desirable not to have

NPQ at all and have somewhat lower growth light, saving on the power in the greenhouse.

Moreover, the metabolic state of plants could vary during the day, stage of development,

and so on that would inevitably reflect in the light saturation requirement and NPQ itself.

Indeed, the rate of CO 2 assimilation would determine the rate of ATP and NADPH

consumption that in turn would affect the levels of reduction of the electron transport chain

and lumen acidification. The latter would control zeaxanthin and in the end the levels of

energy dissipation in antenna via NPQ. Therefore the development of a system of a feed-

back control that finely adjusts the light intensity in greenhouses to the needs of plants

avoiding unwanted saturation effects and wasteful excitation energy losses would be an

ultimate energy-saving measure for the new generation of agriculture.

The fundamental research on improvement of efficiency of energy utilization in

photosynthesis is important not only for yield of crops but for saving space on land and

saving the wild environment from the effects of agricultural activities. The great challenge

is that the ways photosynthesis processes and stages evolved make it very hard to manoeu-

vre their adjustments to bring about fast, effective and reliable crop improvement. One can

argue that instead of trying to find ways to enhance the photosynthetic efficiency, one can

try to manipulate regulation of photosynthesis in the context of plant development and

growth enlarging, for example, the volume of the photosynthetic machinery. Making more

room for it by expanding the total surface of the photosynthetic membrane and the carbon

assimilation machinery is one of the ways of exploring potential enhancement of crop productivity. Indeed, the well-developed canopy of bamboo could be one of the central

factors enabling its very fast growth rate (Figure 10.3 ). One has to explore the great advan-

tage of the nanoscale design of the photosynthetic membrane, remembering Feynmann’s

famous ‘plenty of room at the bottom’, mentioned in the Chapter 2. If not the efficiency of

the photosynthetic machinery, the scale of its design should be exploited to enhance the

productivity of the process. Naturally, there will be some limitations to overcome, arising

from the creation of strong light gradients within the cell, and so on.

10.4 Light Harvesting Principles for Future Applications: Liberation from Saturation Constraints

The principles of light harvesting antenna design come useful when the questions of the

use of alternative energy sources for the needs of mankind arise. The fundamental idea is

that mankind has to learn to live in harmony with the Biosphere. Instead of using the

polluting and anyway nearly exhausted products of the millions of years of photosynthe-

sis, fossil fuels, we must learn to use the most natural source of energy available for our

planet, the Sun. The whole Biosphere uses only a tiny fraction of the Sun’s energy as was

mentioned previously. This means that the problem of energy shortage did never appear

in it until the modern human civilization has evolved. Its progress pushes the boundaries

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of the Biosphere, wiping out species, exhausting its resources and poisoning the environ-

ment. In the end, human activities start to undermine greatly the advantages of living in

the civilized world as we know it. Hence, the need to exist in harmony with the Biosphere

should be the first priority of the modern society. The solution of the energy question is

of paramount importance towards reaching this goal. One hundred thousand TW of avail-

able power from the Sun is an immense number. Currently, human beings use about 14

TW, more than 60% of which comes from burning fossil fuels. The maximum energy that

can be extracted from the other alternative sources like wind or tidal could only partially

satisfy the growing global demand for energy. Nuclear fission is another alternative, but

expensive, potentially dangerous, polluting and cannot be sustained for long, since the

resources of nuclear fuels are limited. Nuclear fusion is much more attractive and power-

ful potential source of energy, however, it is not clear whether the technology can be

practically developed at all.

Solar power is impressive. An hour of the global sunlight energy equals to the yearly

energy needs of the mankind. Solar panels with only 5% efficiency of energy conversion

covering the size of 10% of Sahara desert can produce ~20 TW energy, which again,

sufficient to satisfy the world energy demand. These figures suggest that solar power should

be seriously considered as the major source of energy for our civilization. Moreover, the

demand for energy grows quickly. In the recent 50 years it grew almost three times. The

share of electric power demand is about 60%. And light energy is relatively easy to convert

in the energy of moving electrons, as the nature in the form of photosynthesis has clearly

demonstrated. Indeed, photons interact with matter via electrons, charging them with

energy, that can cause electron removal from molecule and transfer elsewhere, initiating

what is basically an electric current process (see Chapters 3 and 5). In technological reality

common photovoltaic devices can be based upon the use of silica-based materials. They are

semiconductors, that is, materials that possess limited amount of charges that can form the

electric current. An essential parameter of a photovoltaic semiconductor is its band gap:

energy difference between the electrons based in the valence band and electrons that are

based in the conduction band and possessing much higher energy than the valence electrons.

The band gap for silica is 1.12 eV or ~1116 nm, a near infrared region of the solar spectrum

(Figure 10.4 ). Unfortunately this region is not at the maximum of the natural light spectrum

and can be strongly obscured by the water vapour absorption. The efficiency of silica-based

photovoltaic devices can practically reach 20% or slightly more.

Currently the solar energy industry is at the embryonic stage. The whole world consumes

only around 2 GW of Sun power, a tiny 0.01% of the total energy demand and 1/(2 × 10 8 )

fraction of freely available light energy. Why was the vast energy of the Sun not broadly

used and instead we chose fossil fuels: a chemically stored light energy via millions of

years of photosynthesis? The answer is that the science and engineering some 100 years

ago were nowhere near the levels they are now and therefore relatively cheap fossil fuels

were adopted for various energy requirements. Progress in chemistry and chemical

engineering in the nineteenth and twentieth centuries paved the way for mass manufacture

of various types of vehicles and heavy machinery that transformed the world we live in. In

addition, the domestic demands for water, heat and light have been greatly satisfied on a

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colossal scale. At the beginning, nobody thought that the fossil fuels would run out.

Nowadays, it is a relatively accurately predicted reality.

The use of energy of light globally becomes unavoidable. Apart from being freely

available, it will continue to be available for some time, another several billions of years.

The consumption of light energy can be made absolutely non-polluting: an important factor

enabling our civilization to live in harmony with the Biosphere. There is plenty of light

energy available, much more than we would ever need. However, there are also disadvantages

in this form of energy. First of all, it has an intermittent nature: the Sun shines brightly

(when there are no clouds) only during a day. Hence, there is a need to transform and store

its energy in a different form, as photosynthesis does in the dark. It simply makes mole-

cules by fixing CO 2 . The chemical form of energy can be stored for a long time. Therefore,

the solar energy industry has to address the storage problem accordingly. Another challenge

is that any global enterprise, like mass-conversion of all industries for the new energy

carrier, is enormously expensive and time-consuming, particularly at the beginning and

when the traditional forms of energy are still available. Naturally, the old resists the change

brought about by the new. And finally, another disadvantage of using light is that in most

cases it is a very dilute form of energy. Yes, globally it is large, but practically, it requires

collection across the significant space. The evolution of photosynthesis invented the light

harvesting antenna to enable energy concentration within the membrane and for the

photochemical reaction centres. The emergence of antenna made it possible for organisms

to live in very shaded environments. Improving the light capturing efficiency in the solar

devices as well as their photoconversion efficiency could greatly enhance energy input, and

save space and resources.

The combination of fast progress in nanotechnologies with knowledge of the principles

of organization of photosynthetic light harvesting is a promising way to enhance the

efficiency and power of solar devices. An example of such technology can be the titanium

oxide-based photovoltaic devices coupled with a synthetic dyes absorbing infrared or

Wavelength, nm

500

Ligh

t int

ensi

ty O2

visible

Sun’s infrared region

H2O/O2

15001000 2000 2500 3000

H2O

Figure 10.4 Absorption of infrared sunlight by atmospheric water, oxygen and carbon dioxide.

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visible light to overcome the relatively large band gap of titanium oxide (~3.2 eV corre-

sponding to 387 nm). In addition, this band gap can be tuned by the doping of the material

with various elements (N, Sn, Co, etc.) or/and applying various nanotechnologies. These

measures can result in tuning the absorption of the photovoltaic media to the maximum of

the visible light. An additional way to enhance and spectrally tune the photovoltaic media

could be the attachment of LHC antenna complexes. As was presented in Chapters 5–9, the

photosynthetic antenna possesses several remarkable features in its design that could be of

a great advantage in the creation of a new generation of photovoltaic nanostructured cells.

These advantages include a very high absorption cross-section, spectral tenability, relatively

good stability, small size and possibility to regulate the electronic excited energy levels by

tuning the excited state lifetime of the bound pigments. The potential to manipulate and

mutate the antenna protein offers a broad range of modifications of its properties, like

cross-section and attachment to semiconductors in various forms, like films, porous media

and quantum dots which can be largely free from the saturation effects that take place after

the light harvesting step in the chain of photosynthetic events (see Chapter 9). Light energy

collection principles of the natural antenna can also be utilized in the design of artificial

light harvesting media based upon highly absorbing pigments of, for example, the

porphyrin/chlorin family (for structures, see Chapter 5). Nature selected only a few organic

pigments to serve for light harvesting purposes. Hence, chemical modification and assem-

bly of these pigments into light harvesting antennae is the attractive strategy for the creation

of artificial photovoltaic systems. This strategy coupled with the research in nanomaterials

is an attractive future venue in creation of artificial solar energy systems. Chapters 3, 6 and

7 demonstrated the very complex hierarchy of photosynthetic membrane structures, in

particular light harvesting antennae. Indeed, the two-dimensional multisubunit assembly of

antenna around reaction centres is eventually compacted into the three-dimensional

organization of granae membranes carrying PSII. In addition, the arrangement of thylakoid

system within the chloroplast, chloroplasts within the cell and cells within the leaf tissue

create an optimized and regulated light capture process on the macroscopic level. Hence,

the nanoscale design of the photosynthetic antenna enables the most effective way of light

harvesting and, at the same time, photoprotection (see Chapter 9). Moreover, this design

ensures an impressive stability of the photosynthetic membrane made of vulnerable

molecules; lipids, pigments and proteins.

The use of light harvesting principles is not limited to the photovoltaic technologies. The

hydrogen production using solar energy and utilizing the mechanism of water splitting in

the photosystem II is another venue that is being intensively discussed and explored nowa-

days. Hydrogen is an ultimate pure form of energy in comparison to the carbon-based

fuels: the burning of hydrogen produces water as the final product. The efficiency of water

splitting by PSII driven by red light can reach 50%. Recent crystallographic advances ena-

bled researchers to describe the mechanism in very detail. One of the ways to utilize PSII

for the solar energy conversion into hydrogen would be to safely immobilize its complex

on the media that would preserve its vulnerable multisubunit structure. Again, novel devel-

opments in the materials sciences, particularly those researching new media for nanotech-

nologies are required to immobilize PSII for hydrogen production. As far as light harvesting

Rupan_c10.indd 252 8/29/2012 5:34:55 PM


is concerned, LHCII is a natural complex feeding PSII with energy, therefore it can be

naturally integrated within the immobilized system. The antenna size can exceed 300 chlo-

rophylls per one reaction centre with efficiency at about 80%.

In addition to PSII, PSI complex can also be utilized for creation of hybrid solar cells.

Pioneering work on immobilization of PSI complexes on electrodes has already started to

emerge. Being more stable and more efficient than PSII, PSI is a promising complex to be

explored in solar technologies. Its antenna size is smaller than that of the PSI, however, it

has a docking site(s) for LHCII complex and therefore can be ‘equipped’ with the extra

antenna to maximize the power of the photon energy conversion into the energy of moving

electrons. It is important to mention that the photochemistry in the reaction centres of pho-

tosynthesis can also be used for creation of the new types of photosensors or bioindicators.

This venue of applications is not as well-publicized or investigated as the search for alter-

native energy. However, the photosensors or low-energy photo transformation devices

could be very useful in various aspects of our day-to-day life, such as assessing the pollu-

tions in the environment, testing water and food quality, powering various personal elec-

tronic gadgets we cannot live without in the modern society. Finally, the principles of light

energy harvesting and utilization may well become useful in medical applications and, who

knows, maybe in the future the human organism could be adopted to utilize the energy of

the Sun directly, a sci-fi scenario today but tomorrow’s reality? Taking into account the

ultimate ambition of the mankind to conquer the universe, light for space travel may be the

only source of available energy for all needs.

10.5 Effects of Changing Climate – The Onset of Disorder

Unfortunately, the approaching energy crisis is not the only global problem our

civilization has to address in this century. The levels of carbon dioxide in Earth’s

atmosphere have risen by ~28% in the last 300 years, by 17% in the last 100 years and by

~13% in the last 50 years. The progression is steepening up in a good correlation with the

increasing global manufacturing, population and energy demand. There is a little doubt

that mankind is involved in causing the CO 2 increase. Discovery and utilization of fossil

fuels, followed by industrial revolution and the arms race are to be blamed. Nearly

6 billion tons have been emitted into the atmosphere as a result of fossil fuel products

burning. Forecasts predict it will triple by the year 2050 due to the steeply increasing

human population and energy demand.

Despite being still very low (<0.04%), carbon dioxide concentration in the atmosphere

started to impact the global climate. The average global temperature has risen by ~0.6 °C in

the recent 250 years. The increase is believed to be due to mainly carbon dioxide. Figure 10.4

shows the infrared tail of the Sun’s spectrum depicting bands of atmospheric water, oxygen

and carbon dioxide absorption. The latter strongly absorbs in he region of the lowest energy

in the spectrum of Sun. Still, this absorption is trapping vast amount of light energy and

converting it all into heat, causing the global worming effect. Interestingly, that the amount

Rupan_c10.indd 253 8/29/2012 5:34:55 PM


of carbon dioxide in the atmosphere is about 60 times lower than that of the gas dissolved

in the oceans. Global warming would gradually decrease the carbon dioxide solubility in

oceans and cause its release into the atmosphere, accelerating further the global warming

process. Accurate forecasting of the global warming is hard if not impossible since numer-

ous factors affect the process. The general trend, however, remains a great concern of

communities and the governments. The almost explosive on the geological scale global

warming can drastically affect the biosphere and the way we live in it.

Can our knowledge on the photosynthetic membrane be used to help combating or at least

predicting the impact of global warming? As far as the first measure is concerned, the

knowledge of the principles of photosynthesis in development of the alternative sources of

energy as described in the previous paragraph can help, since it would accelerate the aban-

donment of the use of the fossil fuels, hence stopping the further carbon dioxide emissions.

It would remain for us to address and solve the problem of removal of the already emitted

billions of tons of the gas from the atmosphere. This seems to be a titanic task. Recently,

various photocatalysts that reduce CO 2 to methane and other reduced forms of carbon have

been studied. The idea mimics photosynthesis on a simpler, photochemical level. If it works,

the problem of recycling of the carbon dioxide would be solved. Indeed, if we learn the

principles of recycling, so common in the Biosphere, we would embark on the path of living

in harmony with our planet, not disorder, that is detrimental to life. Additionally, our

Biosphere represents a relatively competent carbon carrier, binding nearly 2.6 times more of

this element than it is currently present in the atmosphere. Therefore, the global photosyn-

thesis could be, in theory capable, of doing the job provided that the climatic changes will

not be detrimental to its productivity. The global photosynthesis research in the future would

be very helpful if it addresses the impact of stress factors, such as light, drought and tem-

perature and their combinations on the productivity of the process. Indeed, the knowledge of

the photosynthetic membrane adaptations, described in the Chapter 9 and its broadening to

a multiple abiotic factors would be of a great help in predicting the effect of the global cli-

mate change on the biosphere. Crop sciences informed by this knowledge would enable to

create more resistant crops. Indeed, rapid environmental changes demand rapid measures, a

sign of the onset of a new geological era, the Anthropocene that many scientists have started

to talk about. The science of ocean photosynthesis will become increasingly relevant since

not only the global ocean carries vast amounts of carbon dioxide; it possesses great capacity

for CO 2 reduction. This is being limited by the deficit of iron and other vital for photosyn-

thesis microelements. Currently the aquatic photosynthetic productivity is about 50% of the

yield of land photosynthesis. Improvement of aquatic photosynthesis would also be of great

help for development of algal biodiesel technologies. Growing algae for fuel would not be

limited by space. In addition, it would be quicker and easier to create mutants that possess

high photosynthetic rates compared to land plants.

Bibliography

Barber , J. ( 2007 ) Biological Solar Energy . Phil. Trans. R. Soc. A , 365 , 1007 – 1023 . Blankenship , R. ( 2002 ) Molecular Mechanisms of Photosynthesis . London : Blackwell Science .

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Boeker , E. and van Grondelle , R. ( 1999 ) Environmental Physics . Chichester : John Wiley & Sons, Ltd. Clayton , R.K. ( 1980 ) Photosynthesis. Physical Mechanisms and Chemical Patterns . Cambridge :

Cambridge University Press . Hall , D.O. and Rao , K.K. ( 1995 ) Photosynthesis . Cambridge : Cambridge University Press . Lewis , N.S. and Nocera , D.G. ( 2006 ) Powering the planet: chemical challenges in solar energy

utilization . Proc. Natl. Acad. Sci. U.S.A. , 103 , 15729 – 15735 . Moore , T.A. and Gust , D. ( 1989 ) Mimicking photosynthesis . Science , 244 , 35 – 41 . Murchie , E. and Horton , P. ( 2011 ) Manipulation of photoprotection to improve plant photosynthesis .

Plant Physiology , 155 , 86 – 92 . Murchie , E. , Pinto , M. and Horton , P. ( 2009 ) Agriculture and new challenges for photosynthesis

research . New Phytologist , 181 , 532 – 552 . Nocera , D.G. ( 2012 ) The artificial leaf . Acc. Chem. Res. , 45 , 767 – 776 . Qiu , G.X. , Shen , Y.K. , Li , D.Y. et al . ( 1992 ) Bamboo in sub-tropical eastern China , in: Primary

Productivity of Grass Ecosystems of the Tropics and Subtropics , S.P. Long et al . (eds). London : Chapman and Hall .

Ruban , A.V. , Duffy , C.D.P. and Johnson , M.P. ( 2011 ) Natural light harvesting: principles and environmental trends . Energy and Environmental Science , 4 , 1643 – 1650 .

Vernadsky , V.I. ( 1997 ) The Biosphere: Complete Annotated Edition . Heidelberg : Springer . Walker , D. ( 1992 ) Energy, Plant, Man , Brighton : Oxygraphics . Zhu , X.G. , Long , S.P. and Ort , D.R. ( 2010 ) Improving photosynthetic efficiency for greater yield .

Annual Review of Plant Biology , 61 , 235 – 261 .

Rupan_c10.indd 255 8/29/2012 5:34:55 PM




‘… Will draw the thing as he sees it . For the God of things as they are!’

Rudyard Kipling

The aim of this book was to introduce the reader to the phenomenon of the photosynthetic membrane , the natural nanoscale world which evolved to provide life with its vital

component, energy. The ways in which this structure captures the fairly dilute energy of

light and adapts to its fluctuations were the focus of this text. One of the essential points the

author wanted to make is how the complexity and entropy of this biological system are

being conquered and exploited by the structure in the manner that is central for its chain of

functions: photon-exciton-electron-proton-chemical energy transformations. Despite the

fact that the photosynthetic membrane functions are primarily based upon the presence of

pigments, the central role of protein was explored, underlined and explained through the

examples of how it fulfils the accommodating, conducting and regulatory functions of

the photosynthetic light harvesting process. The outstanding principle that enables these

numerous sequential energy-transduction events is the self-regulation that is largely based

upon the grand structural design of the membrane.

The organization of light harvesting machinery in the two-dimensional space of the

membrane is an outstanding biological phenomenon that allows for a more organized and

controlled functioning of its numerous components. This is just one of the many ways in

which life conquers the inevitable entropy potential of the complexity of its components.

The membrane is well-compacted in the thylakoid system of chloroplasts so that the surface

area is greatly maximized and can be varied in response to the availability of light in the

environment. Amazingly, the world of the membrane is microscopic and yet its total surface

area within the chloroplast and leaf can be very large. The nanoscale organization provides

Conclusions

11

Rupan_c11.indd 257 8/29/2012 5:34:23 PM


an environment with many different physicochemical properties to those of the macroscale.

It is of great advantage to have a modular organization of the membrane components,

specifically the light harvesting antennae. This organization enables the dynamics that

underlies its functions and adaptations, robustness and plasticity and most importantly the

autonomic control of its efficiency.

The photosynthetic membrane is built by a range of forces of which noncovalent ones

are of a great importance. They determine bilayer formation and integrity and membrane

protein secondary, tertiary and quaternary structures. The transient nature of these forces

enables the dynamics of the photosynthetic membrane components which manifest in lipid

and protein diffusion and conformational flexibility. Nanoscale life processes simply float

in an array of various non-covalent interactions, transient and yet very deterministic to the

point that they can accurately reproduce the ‘working’ structures of such vital molecules as

DNA and proteins. In the membrane these forces govern complex relationships between

various types of membrane proteins, stabilizing their macrostructural organization and lat-

eral distribution that are of paramount importance to the membrane functions. With amaz-

ing accuracy the proteins of the photosynthetic membrane handle tiny particles of matter:

photons, electrons and protons. These are millions of times smaller than the protein size

and yet being precisely directed throughout numerous membrane complexes, their

energy transformed and utilized for the creation of chemical bonds: a fundamental

realization of the accumulated negative entropy in the cell.

The most fundamental function of the photosynthetic membrane is to capture sunlight;

hence the most abundant components are the light harvesting pigment-protein complexes.

This book aimed to explain the evolutionary necessity for the emergence of a specialized

group of proteins that bind pigments in order to efficiently intercept photons and concen-

trate their energy into the photosynthetic reaction centre for the primary photochemical

transformation. Effective collection of light – light harvesting – is vitally important; it

significantly broadens plant habitats, allowing them to live in extremely shady environ-

ments. The organization of light harvesting antennae also ensures efficient control of the

excitation pressure that can be built up in high light. Therefore, one of the principles of

antenna organization is its flexibility that stems out of the existence of a feedback control

of its efficiency by the proton gradient. The latter signals a saturation of the energy

transformation events in the membrane and sends a message to antenna to reduce light

harvesting efficiency, so that part of the absorbed light energy can be safely dissipated as

heat. Therefore, fluctuations in light intensity, even on timescale of minutes, are dynamically

tracked within plant leaves without an obvious visible manifestation. It is only when

chlorophyll fluorescence is measured that the highly dynamic nature of the light harvesting

process is revealed. Owing to this the photosynthetic machinery of a leaf can remain intact,

with the pigments green and capable of light harvesting for many days and even months,

whilst isolated natural pigments are generally promptly bleached and damaged by exposure

to light. One of the central properties of the natural light harvesting process is that all

antenna pigments are bound to the protein. The protein serves as a conductor of pigment

functions. It binds them in specific places, determining the distances between them, their

mutual orientations, and their interactions with each other and the environment. The protein

Rupan_c11.indd 258 8/29/2012 5:34:24 PM

Conclusions 259

is therefore a ‘programmed solvent’ that is genetically predetermined and capable of

accommodating the pigments at very high concentration without compromising their

function. It also ensures that the pigments are well-protected against the various unwanted

consequences of excess light, like triplet states, singlet oxygen, radicals, and so on. The

antenna xanthophylls, secondary pigments, are vitally important in protection. However,

this is not the only function of this class of molecules. They are also crucial in ensuring the

structural integrity and correct folding of the light harvesting protein. Hence the nature of

antenna molecular design reveals a cooperative character of the functioning of its structural

elements. This cooperativity is also central to the regulation of a sequence of photosynthetic

reactions that take place in the membrane, ensuring their relatively autonomic character.

The latter is an essential manifestation of life’s way of conquering entropy: the multiple

constituents of a biological system have evolved in such a way that their structure ensures

a smooth assembly into the complex functional cell elements/compartments which interact

with each other and accomplish complex functions of an essentially autonomic nature.

The atomic resolution structures of antenna complexes have just started to emerge and

our understanding of the function of the antenna’s various elements is still at an embryonic

state. However, the atomic resolution of LHCII complexes opened a rich world of vector

(directional) and quantum chemistry and physics of the workings of the molecular light

harvesting machine. This text gives an insight into how it is assembled within the photosys-

tem structure. It also shows numerous examples of the properties and fascinating behaviour

of isolated light harvesting complexes; these nanodevices that evolved to capture light with

the highest efficiency. Classic and emerging approaches in their studies are described here

in order to introduce the reader to the sheer spectrum of multidisciplinary expertise and

experience that have accumulated over several recent decades of the photosynthesis

research. Most exciting is the progress made in the field of visualization of the intact

membrane as well as the work on single proteins. Their success and rapid development

promises to give more insightful and mechanistic information about the workings and life

of the photosynthetic membrane.

It is reasonable to expect that in future new high resolution in vivo visualization and

protein tracking techniques will be developed, giving a direct picture of the behaviour of

individual groups of complexes or even single proteins. Parallel development of molecular

modelling techniques is necessary in order to describe the two-dimensional diffusion and

clustering processes in the intact membrane and the dynamics of individual proteins.

Science of the photosynthetic membrane, when equipped with such tools and approaches,

will eventually become more routinely predictive, giving accurate quantitative information

about the mechanisms of function and adaptation within the complex networks of proteins

and cofactors of the photosynthetic membranes, taking the whole physiology of the

photosynthetic process to a principally new level of understanding. The science of the bio-physical chemistry of live photosynthetic membranes would become a fundamental basis

for the new generations of plant physiologists, who would be able to explain and project the

various consequences of mutagenesis, adaptation strategies and the responses of the

photosynthetic machinery to various environmental factors. The new level of molecular

approaches and knowledge would become crucial to the work directed at improving the

Rupan_c11.indd 259 8/29/2012 5:34:25 PM


productivity of crops and the adaptation strategies made necessary by a changing global

climate. The mechanistic and quantitative character of the new plant science would enable

researchers to accurately assess the productive capacity of various crops and predict the

quality of their adaptive potential. It would also benefit greatly the creation of ecological

models that predict the impact of global warming on plant communities and the biosphere

in general. Research into the physicochemcial principles of light harvesting would also

indicate whether they can realistically be utilized in the development of highly efficient,

stable and cheap solar energy devices. And, last but not least, it should become increasingly

fascinating and educational for us to observe and study the ways in which evolution han-

dled the laws of chemistry and physics in creating the photosynthetic membrane, a system

with form and function so fantastically complex yet so beautifully deterministic: a system

which ultimately sustains life on planet Earth.

Rupan_c11.indd 260 8/29/2012 5:34:25 PM

(a) (b)

(c) (d)

(e) (f)

(g) (h)

Chloroplast

Granae

Granae

Granal thylakoid

PSIILHCII

PSII

Plate 3.1 Origin and organization of the photosynthetic membrane of higher plants. (a) Spinach plants – a common model for mechanistic photosynthesis research. (b) Fluorescence confocal image of chloroplasts in the intact leaf showing clearly separated fluorescing dots – granae. Bar is 5 mm (courtesy of Tomasz Goral and Conrad Mullineaux). (c) Isolated chloroplasts in the incubation medium. Bar is 10 mm. (d) Electron micrograph of a single chloroplast show-ing dark elongated granae and large white starch granulae (centre). Bar is 1 mm. (e) Schematic presentation of thylakoid system structure of the chloroplast. (f) Electron micrograph of a single grana stack (centre). Bar is 200 nm. (g) Freeze-fracture electron micrograph of a single thylakoid membrane from grana showing photosystem II particles. Bar is 100 nm. (h) Freeze-fracture electron micrograph of grana thylakoid membranes displaying photosystem II as well as light harvesting complex particles (LHCII). Bar is 200 nm.

Ruban_bins.indd 1 8/29/2012 9:41:57 PM

Plate 3.2 Scattering and absorption of light by oak leaves. Left: 1, 2 and 3 order of leaves facing light. Right: chlorophyll fluorescence confocal image of the leaf cell showing even redistribution of chloroplasts. Bar is 5 micrometers (Photo on the right is courtesy of Tomasz Goral and Conrad Mullineaux).

Chlorophyll

Lipid

Xanthophyll

Plate 3.5 An example of close van der Waals interactions between a fatty acid residue of phospholipid, chlorophyll phytol tail and xanthophyll. The coordinates were taken from the structure of LHCII complex (for details see Chapter 6).


Leaves Chloroplasts

Membranes

LHCII

Separated complexes

LHCIIb

LHCIIdLHCIIcLHCIIa

0.4

pH

4.2

4.6

0.7

Sucrose,M

PSII

PSI

Membrane fragments andsolubilised complexes

Grinding &centrifugation

Osmotic shock &centrifugation

+ Detergent &centrifugation

Ultracentrifugation onsucrose gradient

Isoelectric focusing(IEF)

Plate 4.1 Basic steps of isolation of the photosynthetic membrane complexes.


Plate 5.10 Global redistribution of chlorophyll a. Reproduced with permission from http://oceancolor.gsfc.nasa.gov/SeaWiFS/. © SeaWiFS Project/NASA.

Plate 6.5 Chlorophylls of LHCIIb monomer in 2.72 Å resolution structure (left) and tight circular way of chlorophyll arrangement around the hydrophobic core of the complex (right). Presented in freeware PyMol 0.99.


Plate 6.6 Chlorophylls a of LHCIIb monomer. Arrangement around the apoprotein (top), geometry and nomenclature (middle) and types of ligands (bottom). Ph: phospholipid. Presented in freeware PyMol 0.99.


Plate 6.7 Chlorophylls b of the LHCIIb monomer. Arrangement around the apoprotein (top), geometry and nomenclature (middle) and types of ligands (bottom). Presented in freeware PyMol 0.99.


Plate 6.8 Structure of neoxanthin-binding domain of LHCIIb showing enrichment in Chl b (left) and 9-cis end of the molecule hydrogen bonded to tyrosine Y112 (right) in the vicinity of the antiparallel strand of the lumenal loop near helices C and E. Presented in freeware PyMol 0.99.

Plate 6.9 Structure of lutein 620-binding domain of LHCIIb showing the terminal emitter chlorophylls a610-612 (top) and hydrogen bonding patterns to hydroxyl oxygens of the xanthophyll (bottom). Presented in freeware PyMol 0.99.


Plate 6.10 Structure of lutein 621-binding domain showing the domain chlorophylls a602-604 (top left), hydrogen bonding patterns to hydroxyl oxygens of the xanthophyll (top right) and interaction with Chl a603 localized on the neighbouring LHCIIb monomer (bottom). Presented in freeware PyMol 0.99.


Plate 6.11 Structure of violaxanthin-binding domain of LHCIIb showing interactions with chlorophylls a 613-614 (top left), Chl b601 (top right). Below are shown the side (left) and top (right) views of all LHCIIb xanthophylls arrangement within the complex. Presented in freeware PyMol 0.99.


Plate 6.14 Structure of CP29 (LHCIIa) complex at 2.8 Å resolution. Top: side and top views of the whole complex; middle: all xanthophylls view; bottom: the putative terminal emitter chlorophylls a. Presented in freeware PyMol 0.99.


Plate 8.12 Spectra of the sun light and energy absorbed by the LHCII complex in trimeric and monomeric states. Bottom panel shows the relative leaf penetration depth of the sun light of different spectral regions/colour.


0.8 ns 1.1 ns

864Time, ns

Flu

ores

cenc

e, r

el

200

1000

2000

LHCII trimer

LHCII crystal

Plate 8.18 Quenching of chlorophyll a fluorescence lifetime in a single LHCII crystal using FLIM technique. The image is shown in false colours defining the fluorescence lifetime as presented by the colour lifetime scale below the image. Bottom: average fluorescence decay profiles for trimers and crystals of LHCII. Reprinted with permission from Macmillan Ltd Publishers, Pascal et al .© 2005 Nature Publishing Group.


Plant adaptations to light

Number ofmembranes

Size of antenna

ChloroplastmovementsChloroplast

number

Plantmetabolism

Chloroplastmetabolism

Photosystems ratioState transitions

NPQ

Leaf orientationLeaf reflectance

Salt depositsLeaf structure

Managing light absorption

Managing absorbed energy

Plate 9.2 Multilevel strategies of plant adaptations to the light environment. Type A: managing light absorption; type B: managing absorbed energy. Three levels of adaptations are presented: organismal (plant), cellular (chloroplasts) and molecular (photosynthetic membrane). Reprinted with permission from Ruban, A.V.© 2008, Landes Bioscience.

De-epoxidation

Dark / low light Excess light

Δ pH

PSII minor antenna

LHCII trimer

PSII core

Zeaxanthin

Violaxanthin

LHCII aggregate(quenched)

Plate 9.20 Scheme depicting the current knowledge regarding the structural reorganization of the PSII–LHCII macrostructure occurring in the NPQ state. Dissociation of the part of light harvesting antenna from PSII supercomplex leads to the reorganization and aggregation of LHCII complexes. Reproduced with permission from Johnson et al .© 2011 American Society of Plant Biologists.


Plate 10.3 Phyllostachys pubescens, the efficient light harvester and one of the fastest growing plants. Kyoto Rakusai Bamboo Park. Courtesy of Kateryna Law.





Index

absorption cross-section, 74, 82, 177

abstract thinking, 241

acclimative modulation of antenna

size, 201

aggregation hypothesis, 225

Agrobacterium tumefaciens, 40

allene group, 87

allosteric control, 222

9-aminoacrydine, 63, 215

amphiphilicity, 9

anisotropy, 49

antenna cross-section, 94

antenna hydrophobicity, 230

antheraxanthin, 87

Anthropocene, 254

antimycin A, 218

antiparallel strands, 117

antisense, 136

anti-Stokes energy transfer, 97

Archaea, 11

artificial light harvesting, 252

ATP-synthase, 26, 28

attenuated total reflection (ATR), 54

bacteriochlorophyll, 131

band gap, 82, 250

basic natural sciences, 242

BBY particles, 35

Beer-Lambert law, 49

benzoquinone, 24

binary reaction, 191

bioindicators, 253

biophysical chemistry, 259

Biosphere, 4, 241

BODIPY, 30

Boltzmann constant, 4

Bragg’s law, 48

Brownian motion, 8

cantilever, 45

carbon dioxide, 253

β -carotene, 37

carotenoids, 37

cations, 19

cDNA hybridization, 40

charge recombination, 236

charge transfer, 183

chemical storage, 6

chlorin ring, 21, 82

chlorophyll, 24, 37

a/b ratio, 102

b fluorescence, 164

pigments, 82

spectral forms, 87

chlorophyll-chlorophyll associates, 182

chlorophyll-xanthophyll interactions, 183

chloroplast, 17

cholate, 36, 102

9-cis conformation, 115

classification of antennae, 91

classification of membrane proteins, 15

climate change, 253

coarse-grained model, 167–8

coherence, 80

collective dynamics, 14

compartmentalisation, 9

Rupan_bindex.indd 261 8/29/2012 5:40:56 PM

262 Index

concentration quenching, 78, 132

conduction band, 250

configurational alteration, 183

conformational states, 188

Confucius, 5, 33, 67, 101, 197

conical intersection, 235

conjugated double bonds, 24, 87

conjugation, 82

cooperative nature of qE, 233

co-ordination interactions, 119

CP24, 26, 106

CP26, 26, 106, 176

CP29, 26, 106, 126, 128

CP43, 26

CP47, 26

CPa, 105

critical micelle concentration

(cmc), 179, 190

crops, 247

cross-linkers, 42, 141, 218, 234

cross-section, 132

cryoelectron microscopy, 149

cryofixation, 44

crystallography, 45, 47–8, 102, 105,

107, 109

C2S2M2L2 particle, 142

C2S2M2 particle, 142–3, 147

C2S2 particle, 140

Cuscuta reflexa, 115

cytochrome b6/f complex, 26

damping, 42

Davidov’s splitting, 80

DCMU 61, 216

DEAE, 36

de Broglie dualism, 70

decay-associated spectra (DAS), 166–7

de-epoxidase, 129, 219

delocalized excitonic states, 235

delta-pH ( Δ pH), 28

denaturation, 37, 185

de-phasing energy, 235

deriphat, 106, 138

derivative method, 162

detergents, 24

Dexter electron exchange, 80

dichlorophenol indophenol (DCIP), 52

dicyclohexylcarbodiimide (DCCD), 218

diethyl sulphate, 40

diffractometer, 48

digalactosyl diacylglycerol

(DGDG), 115

digitonin, 35, 102, 138, 149

dipole moment, 71

dodecyl- α -D-maltoside, 139

dodecylglucposide, 106

dodecylmaltoside, 138

D1 protein, 27, 208

D2 protein, 27

EFs, 140

electrochemical potential, 15

electrochromic shift (ECS), 53, 168

electromagnetic wave, 68

electron

diffraction, 48

exchange, 81

transport, 6, 15

electrophoresis, 36

energy

balance, 243

imbalance (IB), 204

losses, 243

migration, 80

redistribution, 208

transfer rate, 109

transformations, 257

enthalpy, 185

entropically-driven, 185, 188

entropy, 2, 119, 242

negative, 2, 67

environmental changes, 254

enzymes, 16

epoxidaze, 219

Escherichia coli (E. coli) 38, 40

evanescent wave, 42

evergreen plants, 226

exchange interaction, 237

excitation fluorescence, 56, 206

excited state lifetime, 163

excitonic coupling, 79, 235

interactions, 237


Index 263

exciton transfer integral, 235

extinction coefficient, 50

fatty acids, 23

feedback control, 215

ferredoxin, 28, 52

Feynman, 7

ficoll, 40

fluorescence

band F700, 181

confocal, 41

damping, 42

excitation spectrum, 94

lifetime imaging (FLIM), 186

lifetime spectroscopy (TCSPC)

polarisation, 58

recovery after photobleaching (FRAP),

30, 42, 148, 223, 227

time-resolved, 58

yield, 59

Fo quenching, 218

forward genetics, 40

Fourier transformation, 48

FPLC, 35

Frank-Condon principle, 73

Fraunhofer lines, 70

free pigment zone, 105

Förster’s theory, 76

gallium phosphide, 41

gammaaminobutyric acid (GABA)

gel filtration, 35, 127, 140

global analysis, 167

global warming, 242, 254

glucose, 243

glucosides, 24

glutaraldehyde, 185

G-protein coupled receptors (GPCR)

grana stacking, 19, 104

harmful excitation energy, 198

heat emission, 218

heterotrimers, 136

higher occupied molecular orbital

(HOMO), 72

high hydrostatic pressure, 185

His-tag, 185

homogenous broadening, 72

homotrimers, 136

HPLC, 37

H-type aggregate, 89

hybrid solar cells, 253

hydrogen

bond, 10

bonding patterns, 183

production, 252

hydrophilic, 9

resins, 180

hyperbolic decay, 191

hysteresis, 223

icosahedral spherical particles, 115

icosienamer, 143

incoherent coupling, 237

inductive resonance, 81

inhomogeneous broadening, 72

inorganic deposits, 199

integral membrane-spanning, 13

internal conversion, 74

ion channels, 16

ion-exchange chromatography, 127

ionic interactions, 139

isoelectric focusing (IEF), 36, 190

isoenergetic, 185

isoprenoid residues, 23

J-type aggregate, 89

kanamycin-resistance, 40

kinase, 207

Kipling, 17, 267

knock-out mutations, 136

laurate, 10

leaf orientation, 199

LH2, 129

Lhcb2 antisense, 150, 209

Lhcb5 antisense, 141

LHCII, 26, 105–6, 115

crystals, 186

oligomerization, 179

LHCP, 105


264 Index

life, 1

light

adaptations, 198

capture, 6

energy accumulation processes, 6

energy losses, 244

harvesting gene family, 136

harvesting principles, 15, 252

saturation, 198

starvation, 198

lipid, 21

bilayer membrane, 9

mobility, 30–31

liposomes, 39

lithium dodecylsulphate (LDS), 38

Longfellow, 241

long-term adaptations, 201

lowest unoccupied molecular orbital

(LUMO) 72

low-temperature fluorescence, 181

lumen, 19

lutein, 37, 87, 111, 122, 171, 193–4, 209,

235, 246

macrostructural organisation, 135

macroworld, 8

magnesium, 19, 103, 181

managing

absorbed energy, 198

light absorption, 198

mankind, 241

matter, 3

membrane

electric potential, 11

fluidity, 11

permeability, 11

protein, 11–12

metabolism, 15

methanesulfonate, 40

microscopy

atomic force (AFM), 45

confocal scanning, 17

electron (EM), 19, 42

fluorescence, 41

freeze-fracture (FFEM), 13–14, 27,

44–5, 137–8, 140, 147–8, 223–8

optical, 41

microtome, 44

Milne, 136, 169

minor antenna, 106

molar extinction, 74

molecular dimer, 235

monochromator, 56

monoexponential decay, 165

mutagenesis, 40

NADP reductase, 28

nano-materials, 252

nanoscale organisation, 257

nanoworld, 7

NASA satellite, 82

n-body modelling, 14

n-dodecyl- β -D-maltoside, 35–6

negative stain, 227

negative staining, 42

neoxanthin, 37, 87, 111, 121, 171,

193–4

Ni-containing column, 185

nigericin, 216

n-octyl- β -D-glucoside, 35

noncovalent bonds, 258

non-denaturing, 36

non-equilibrium thermodynamics, 119

non-invasive, 159

nonphotochemical chlorophyll

fluorescence quenching (NPQ), 62–3,

214, 248

nonradiative

decay, 59

dissipation, 182

processes, 56, 63

npq4 mutant, 230

N-terminal trimerization motif, 136

nuclear reorganization energy, 73

numerical aperture, 41

octylglucoside, 38, 106, 138

opal glass, 51

optical density, 50

order, 1

oscillating electric dipole, 68

oscillator strength, 74

overlap integral, 77

Ovid, 7


Index 265

oxygen evolving complex, 27

oxygenic photosynthesis, 197

parameters of light harvesting antenna, 94

percoll, 34

PFs surface, 140

Δ pH, 64, 215

Phaeodactylum, 221

phaeophytin, 87

phosphatase, 207

phosphatidylglycerol (PG), 117, 126

phosphodiester, 117

phospho-LHCII, 207

phospholipase, 177

phosphorylation of LHCII, 205

photochemical quenching (qP) 62–3

photoconversion efficiency, 251

photoelectric effect, 70

photoinhibition, 214

photoprotective efficiency, 248

photoreceptors, 200

photosensors, 253

photosynthesis, 4

photosynthetic

control, 215

saturation, 198

unit, 76, 135

photosynthetically active radiation

(PAR), 245

photosystem

I (PSI), 26

II (PSII), 26

photosystems ratio, 200–201

photovoltaic devises, 250

pH titrations, 191

Phyllostachys pubescens, 247–8

phytol, 22

pigment stoichiometry, 106

pK, 233

Planck constant, 70

plasticity of antenna, 176

plastocyanine, 29

plastoquinone 24, 207

polarisation, 53

polyacrylamide gel electrophoresis

(PAAG), 36, 102

polymerase chain reaction (PCR), 40

population growth, 242

porins, 16

porters, 16

potassium

cations, 19, 103

ferricyanide, 52

principles of light harvesting, 93

programmed solvent concept, 93, 109,

164, 179, 181

protein

landscapes, 147

mobility, 30–31

proteolysis of LHCII, 201

proton

binding equilibrium, 233

transport, 6

proton-antenna association constant, 222

PsaA, 26

PsaB, 26

PsbA, 26

PsbB, 26

PsbS protein, 218, 223, 248

PSI

cross-section, 207

supercomplex, 145

PSII

supercomplex, 140

turn-over, 29

yield, 220

pulvinar motor tissue, 199

purple bacteria, 24, 129

qE quencher, 234

quenching

in gel, 185

kinetics, 190

quinone, 23–4

radiative

energy dissipation, 74

lifetime, 59, 74

radical

cation, 236

pair, 214

Rayleigh scattering, 54

receptors, 16

recombinant protein, 37, 111


266 Index

recycling principle, 1

red energy traps, 96

Redfield theory, 80

redox state, 208

reflectance, 51

refractive index, 89

reorganisation energy, 73

resonance Raman, 113

respiration proceses, 244

reverse genetics, 40

Rhodobacter sphaeroides, 129

rhodopin glucoside, 24, 131

Rieske iron-sulphur protein, 28

robustness of antenna, 150

Rodopseudomonas sphaeroides, 24

Roman Empire, 12

rotations, 72

scattering, 51

second derivative, 172

selective light scattering, 182

selectivity, 159

self-regulation, 257

senescence, 226

short-term adaptations, 202

signalling, 15

Snowball Earth, 2

sodium dodecyl sulphate (SDS), 36, 102, 138

solar

energy capture, 242

panels, 250

power, 250

Soret band, 160, 177–8

spectroscopy, 49, 159

absorption, 49, 160

circular dichroism (CD), 39, 43, 160,

163

fluorescence, 56, 160

infrared (IR), 54

linear dichroism (LD), 53, 160, 162

pulse amplitude modulated (PAM), 59

Raman, 54, 171, 183–4, 188

single molecule, 188

Stark, 173

time-correlated single photon counting

(TCSPC), 59, 193

time-resolved, 165

transient absorption, 169–70, 183

spillover, 103, 207

spontaneous fluorescence quenching, 190

stacked thylakoids, 98

stacking, 19

state I, 203

state II, 203

state transitions, 202

steroid structure, 138

stimulated emission depletion (STED), 42

Stokes shift, 72, 95

stray light, 51

sunflecks, 202

supercomplex, 14

Superdex 200 column, 35

symmetry-forbidden, 183

T-DNA insertional mutagenesis, 40

terminal emitter, 183, 246

terpenoid, 24

tertiary

amines, 218

structure, 112, 119

thermodynamic equilibrium, 2

thin layer chromatography (TLC), 38

thin section, 229

thylakoid, 19

titanium oxide, 251

tocopherol, 23–4

total internal reflection (TURF), 42

Tower of London, 11

trans-cis isomerisation, 24

transgenic, 40

transition dipole moment, 74

translational movement, 72, 162

transmembrane helixes, 109

transport, 15

trimerisation, 126

trimers of CP26, 176

triplet excited state, 75

triton X100, 35, 103, 138

turn-over rate, 246

ultracentrifugation, 35

universe, 2


Index 267

unstacked thylakoids, 98

uranyl

acetate, 43

formeate, 43

valence band, 250

van der Waals, 10

vector chemistry, 31

vibrations, 72

vibronic satellite, 94, 163

violaxanthin, 37, 87, 111, 125, 171, 188,

190–191, 193–4, 219, 235

wagging vibrational mode, 113

water splitting, 252

western blot, 37

wonder reflex, 241

xanthophyll, 24, 87

cycle, 219

hydrophobicity parameter

(H-partameter), 194

solubility, 192

zeaxanthin, 37, 87, 123, 129, 171, 188,

190–191, 193–4, 219, 235

zwittergent-16, 146

zwitterionic detergents, 36


the photosynthetic membrane: molecular mechanisms and biophysics of light harvesting

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