3

Photosynthetic water oxidation: The function

of two extrinsic proteins

Tatiana Shutova

Umeå Plant Science Centre

Department of Plant Physiology

Umeå University

Sweden 2007

© Tatiana Shutova, 2007 Umeå Plant Science Centre Department of Plant Physiology Umeå University SE-901 87 Umeå Sweden ISBN: 978-91-7264-481-6 Printed by Arkitektkopia, Umeå, 2007 Front cover photo by Dmitry Belyanin

Abstract

The solar energy accumulated by photosynthesis over billions of years is the

sole source of energy available on Earth. Photosystem II (PSII) uses the

sunlight to split water, an energetically unfavorable reaction where electrons

and protons are extracted from water and oxygen is released as a by-

product. Understanding this process is crucial for the future development of

clean, renewable and unlimited energy sources, which can use sunlight to

split water and produce hydrogen and electricity. In order to do so we need

to understand how this is solved in plants.

I have been focusing on the role of two lumenal proteins associated with the

thylakoid membrane PsbO and Cah3, in the water oxidation process.

Convincing evidences have been presented supporting the hypothesis that

bicarbonate acts as a proton acceptor in the water splitting process in PSII

and the lumenal carbonic anhydrase, Cah3, supplies bicarbonate required for

this function. The PsbO protein, an important constituent of the water-

oxidizing complex, however, its function is still unknown. The PsbO protein

undergoes a pH dependent conformational change that in turn influences its

capacity to bind calcium and manganese, forming a catalytic Mn4Ca cluster

in PSII. We propose that light-induced structural dynamics of the PsbO is of

functional relevance for the regulation of proton release and for forming a

proton sensing - proton transporting pathway. The cluster of conserved

glutamic and aspartic acid residues in the PsbO protein acts as buffering

antennae providing efficient acceptors of protons derived from substrate

water molecules. Both proteins, Cah3 and PsbO have a conserved S-S

bridge, required for proper folding and activity; therefore they are potential

targets for red-ox regulation in lumen.

Sammanfattning

Solenergi som omvandlats av fotosyntesen under miljarder av år är basen

för nästan all energi på jorden. Fotosystem 2 använder solljuset till att

oxidera vatten, ur energisynpunkt en ofördelaktig process, där elektroner

och protoner extraheras från vattenmolekyler vilket ger upphov till syrgas

som biprodukt. Förståelsen av denna process är viktig för att vi i framtiden

skall kunna utveckla rena och förnyelsebara energislag i obegrensad mängd.

Genom att efterlikna fotosyntesprocessen skulle vi i framtiden kunna

utvecka artificiella system som använder solljuset till att sönderdela vatten

för att producera vätgas eller elektrisitet. För att kunna göra det så måste vi

kunna förstå hur dessa processer fungerar i växterna.

Min forskning har fokuserat på att förstå funktionen hos två av de proteiner,

PsbO och Cah3, som deltar i sönderdelningen av vatten. Jag har visat, för

första gången, att ett lumen karboanhydras, Cah3, deltar i regleringen av

den process där vatten spjälkas. Jag postulerar att Cah3 underlättar bort

transporten av protoner från det vattenoxiderande komplexet genom att

generera bikarbonat lokalt, som kan fungera som proton transportör. PsbO

proteinet genomgår en pH beroende konformationsförändring vilket

påverkar dess kapacitet and binda calcium och mangan som i sin tur formar

ett katalytiskt Mn4Ca center i fotosystem 2. Jag föreslår att en ljusberoende

strukturförändring av Psbo är av funktionell betydelse för regleringen av

protonfrigörandet och formar ett proton-avkännande och proton-

transporterande system. Ett kluster av konserverande glutamat- och

aspartat-aminosyror i PsbO proteinet fungerar som ett buffrande nätverk för

protoner som frigörs vid oxidering av vatten. Båda dessa proteiner

innerhåller S-S bryggor ock kan därför vara red-ox reglerade i lumen.

6

List of papers

The thesis is based on the following papers, which will be referred to in the text by their Roman numerals.

I. Villarejo, A., Shutova, T., Moskvin, O., Klimov, V., Samuelsson, G. (2002): A Photosystem II-associated carbonic anhydrase regulates the efficiency of photosynthetic oxygen evolution, EMBO J, 21, 8, 1930-1938.

II. Shutova T., Kenneweg H., Buchta J., Nikitina J., Terentyev V., Chernyshov S., Andersson B., Allakhverdiev S.I., Klimov V.V., Dau H., Junge W. and Samuelsson G. The PSII-associated Cah3 in Chlamydomonas enhances the O2 evolution rate by proton removal, EMBO J, under revision.

III. Shutova, T., Klimov, V., Andersson, B. and Samuelsson, G. (2006) A cluster of carboxylic groups in PsbO protein is involved in proton transfer from the water oxidizing complex of Photosystem II. Biochim. Biophys. Acta (Bioenergetics) 1767, 434-440.

IV. Nikitina J., Shutova T., Melnik B., Chernyshov S., Marchenkov V., Semisotnov G., Klimov V.V. and Samuelsson G. PsbO protein of photosystem II: overexpression in Escherichia coli and the importance of a single disulfide bond for the protein structure stability, manuscript

V. Shutova T, Nikitina J, Deikus G, Andersson B, Klimov V, Samuelsson G. (2005) Structural dynamics of the manganese-stabilizing protein - Effect of pH, calcium, and manganese. Biochemistry 44, 15182-15192

VI. Lundin B., Thuswaldner S., Shutova T., Eshaghi S., Samuelsson G., Barber J., Andersson B. and Spetea G. (2006) Subsequent events to GTP binding by the plant PsbO protein: Structural changes, GTP hydrolysis and dissociation from the photosystem II complex. Biochim. Biophys. Acta (Bioenergetics) 1767, 500-508.

7

VII. Shutova, T., Villarejo, A., Zietz, B., Klimov, V., Gillbro, T., Samuelsson, G

and Renger, G. (2003): Comparative studies on the properties of the extrinsic manganese-stabilizing protein from higher plants and of a synthetic peptide of its C-terminus, Biochim. Biophys. Acta (Bioenergetics) 1604, 95-104.

Paper that is not included in the thesis.

Moskvin, O.V., Shutova, T.V., Khristin, M.S., Ignatova, L.K., Villarejo, A., Samuelsson, G., Klimov, V.V., Ivanov, B.N. (2004): Carbonic anhydrase activities in pea thylakoids - A Photosystem II core complex-associated carbonic anhydrase, Photosynth. Res. 79, 93-100.

8

Abbreviations

BBY – PSII enriched membrane fragments

BC – bicarbonate

CA – carbonic anhydrase

Cah3 – α−carbonic anhydrase, cah3 gene product

CCG – cluster of carboxylic residues

CCM – carbon concentration mechanism

Chlammy – Chlamydomonas reinhardtii

Ci – inorganic carbon: CO2, HCO3- or CO3

2-

DCBQ - 2,6-dichloro-p-benzoquinone; electron acceptor in PSII

DCPIP - 2,6-dichlorophenolindophenol; electron acceptor in PSII

DF - delayed chlorophyll fluorescence

DPC - 2,2'-diphenylcarbonic dihydrazide; electron donor in PSII

E. coli – Escherichia coli

EZ – ethoxyzolamide, a specific inhibitor of carbonic anhydrase

MSP – manganese stabilizing protein

NR - neutral red

PsbO – psbO gene product

PSII – Photosystem II

SDS-PAGE – SDS-polyacrilamide gel electrophoresis

WOC (OEC) – water oxidizing complex

9

Contents

Preface ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ 10

I. Introduction

1. Chloroplast ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ 11

2. Photosystem II structure and function ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ 12

2.1. Membrane proteins and electron transfer steps ▪ ▪ ▪ ▪ ▪ 13

2.2. Extrinsic proteins ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ 14

2.3. Mechanism of photosynthetic water oxidation ▪ ▪ ▪ ▪ ▪ ▪ 15

2.4. Role of calcium in PSII ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ 18

2.5. Role of bicarbonate in PSII ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ 19

3. The Cah3 protein▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ 21

4. The PsbO protein▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ 22

5. Red-ox regulation in lumen ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ 26

II. Results

1. The lumenal carbonic anhydrase is required for functioning and

stability of the electron donor side of PSII▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ 27

2. Cah3 and the proton removing from the WOC ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ 30

3. Cah3 protein is associated with PSII ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ 31

4. Relevance to the PSII from the other organisms ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ 32

5. PsbO and the proton removing from the WOC▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ 33

6. Structural dynamics of the PsbO is necessary for its function,

possible GTP binding▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ 35

7. Red-ox regulation of the PsbO and Cah3 proteins ▪ ▪ ▪ ▪ ▪ ▪ ▪ 38

8. The comparative analysis of two lumenal proteins: PsbO and

Cah3▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ 40

III. Conclusions and outlook ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ 41

IV. Acknowledgement ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ 42

V. References ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ 43

10

Preface

Photosynthesis on the Earth

One of the most important events in the Earth's history occurred when

photosynthetic organisms able to split water were evolved. Accumulation of

oxygen, released as a by-product, resulted in an aerobic atmosphere.

Formation of an ozone layer allowed organisms to move from the ocean to

the land. The solar energy accumulated by photosynthesis over billions of

years is the sole source of all energy available on Earth that has been stored

in the form of coal, oil and gas.

These fuels have been intensively used and are becoming limited. The global

energy consumption, however, will increase from current level of 12.8 TW to

27 TW by 2050 (Lewis & Nocera, 2006), which in turn will lead to a global

warming on the Earth. Therefore, renewable and clean energy sources have

to be developed in the near future. Photosynthesis provides a successful

example of how solar energy can be converted into fuel when electrons are

extracted from water with only light as an energy input. If we can

understand in detail this basic biological reaction, then we will be able to

mimic it for clean and unlimited solar energy utilization.

11

I. Introduction

1. Chloroplast

Photosynthesis is the photochemical process by which plants, algae and

cyanobacteria use the energy of light (photo) for synthesis of organic

molecules (synthesis) and includes light driven electron transport steps as

well as dark reactions. In eukaryotic organisms, photosynthesis takes place

in a small organelle called chloroplast (Figure 1,A). The chloroplast is

enclosed by a double membrane composed of an outer and an inner

envelope. According to the endosymbiosis theory(McFadden, 1999), the

outer envelope has eukaryotic origin, while the inner one derives from an

ancient prokaryote.

A. B.

Figure 1. A. Electron microscopy picture of a higher plant chloroplast. B. Light microscope image of the Chlamydomonas reinhardtii cells.

The chloroplast is filled by a gel-like matrix, called the stroma. The internal

membrane system of the chloroplast, called thylakoids, is organized into

stacked and unstacked regions (grana and stroma thylakoids, respectively)

and encloses a space known as the lumen. The thylakoid membranes are the

site of the photosynthetic light reactions - electron transport chain, resulting

ultimately in the formation of ATP and reduced NADP+. The enzymes

situated in the stroma use the ATP, produced in the light reactions, to store

the trapped energy of light in carbon-carbon bonds of carbohydrates.

12

Chlamydomonas reinhardtii is an unicellular soil-dwelling green algae that

mainly consists of one large chloroplast (Figure 1, B). C. reinhardtii is a

useful model system in photosynthesis research because it combines the

important property of higher plant photosynthesis with the advantages of an

unicellular organism, namely it can grow in the dark on an organic carbon

source while still maintaining a functional photosynthetic apparatus

(Rochaix, 2002).

2. Photosystem II structure and function

Photosynthetic water oxidation, responsible for the production of all O2 in the

biosphere, takes place at Photosystem II (PSII) in the thylakoid membrane

of the chloroplast. PS II is a multi-protein complex composed of more than

25 trans-membrane and extrinsic proteins, a structure that was recently

resolved to 3.0(Loll et al, 2005)-3.5(Ferreira et al, 2004) Å by using X-ray

diffraction. Resolving the PSII structure was an important step forward to

understand the location of cofactors involved in electron transfer within PSII

and the organization of the surrounding protein matrix (Figure 2).

Figure 2. View of the PSII dimer perpendicular to the membrane normal. Helices are represented as cylinders with D1 in yellow; D2 in orange; CP47 in red; CP43 in green; cyt b559 in wine red. The extrinsic proteins are PsbO in blue, PsbU in magenta, and PsbV in cyan. Chlorophylls are green, ß-carotenes are in orange, heme and nonheme Fe are in red, QA and QB are purple. (Ferreira et al.(Ferreira et al, 2004)).

B

13

2.1. Membrane proteins and electron transfer steps

The major red-ox components of PSII are located in the D1/D2 heterodimer

that is composed of 10 transmembrane α helices of the highly conserved

PsbA (D1) and PsbD (D2) proteins. The proximal light harvesting antenna is

located on the CP43 (PsbC) and CP47 (PsbB) proteins. After absorption of a

photon, antennae chlorophylls rapidly transfer excitation energy toward the

reaction centre chlorophyll a dimer, P680. It raises the energy of one of its π

electrons and creates an excited state P680* that is followed by an ultra fast

charge separation and formation of the P680+Pheo– pair, where Pheo is

pheophytin, and P680+● is a cation radical, the strongest biological oxidant

known (E1/2 = 1.2 V). This reaction is considered the highest-energy reaction

in living organisms(Barber, 2003), (Nelson & Yocum, 2006).

On a timescale of about 200 ps, the electron is transferred from the reduced

Pheo- to a quinone molecule, QA. Formation of the QA−P680+ radical pair is

followed by electron donation to P680+ from a nearby red-ox active tyrosine,

called Yz. Yz is a fully conserved Tyr161 residue of the PsbA protein, an

intermediate electron carrier between P680 and the water oxidizing complex,

WOC, where it extracts electrons from a Mn4Ca cluster that, in turn,

removes electrons from two substrate water molecules. YZ accomplishes the

coupling between the reduction of P680+, a one electron transfer, and the

four electron reaction of water oxidation. The red-ox potential of the YZ•

radical matches that of the Mn4Ca cluster even when the Mn4Ca cluster

accumulates oxidizing equivalents. In the time frame of a few ms, the

electron is transferred from QA to the second quinone acceptor in PSII - QB.

When Q

B

B is fully reduced, it becomes protonated and replaced by a

plastoquinone, PQ, molecule from the PQ pool in the thylakoid membrane.

Cytochrome b559 (PsbE and PsbF subunits) is closely associated with the

D1/D2 heterodimer, and may be involved in protection of PSII from

photodamage. In addition, there are a few small (<10 kDa) hydrophobic

peptides consisting of one transmembrane helix that are required for the

assembly and stability of the PSII supercomplex. All red-ox cofactors of PSII

14

bound to the D1/D2 hetero-dimer are precisely orientated in the protein

matrix in such a way that electron flow works with maximal efficiency and

occurs across the thylakoid membrane with the electron donor located on

the lumen side and an electron acceptor located on the stroma side of the

thylakoid membrane (for a recent review see (Nelson & Yocum, 2006;

Renger, 2007).

2.2. Extrinsic proteins

There are three extrinsic proteins associated with PSII on the lumenal side

of the thylakoid membrane in plants and green algae: PsbO, PsbP and PsbQ

with molecular masses of 33, 24 and 18 kDa, respectively. PsbO is present

in all known oxyphototrophs (De Las Rivas et al, 2004). PsbP and PsbQ are

not present in cyanobacteria and red algae, but instead these organisms

contain the corresponding PsbV and PsbU proteins. The PsbO is often termed

the manganese-stabilizing protein (MSP), because in the absence of this

protein, manganese is released from the WOC (Gounaris et al, 1988). The

PsbO protein is highly conserved among higher plant species. However, the

similarity between cyanobacteria and higher plants is only about 50% due to

several deletions and insertions in the protein sequence (De Las Rivas &

Barber, 2004). All oxygenic photosynthetic organisms sequenced contain

one or two copies of the psbO gene. For instance, Arabidopsis thaliana

contains two of them: psbO-1 (At5g66570) and psbO-2 (At3g50820). Both

of these genes are normally expressed, but the PsbO-1 protein is more

abundant under physiologically optimal conditions (Yi et al, 2005).

It is generally accepted that the extrinsic proteins create an optimal

environment (dielectric constant est.) for the water splitting reaction. For

example, intact PSII is not sensitive to addition of a divalent cation chelator;

however, in the absence of PsbP and PsbQ, the PSII activity is strongly

inhibited by this treatment indicating that extrinsic proteins have a

protective function within the WOC (for a review on extrinsic proteins see

(Seidler, 1996). Another role of lumenal proteins in PSII is the controlled

15

transport of substrate water molecules to WOC (Hillier et al, 2001) as well as

transfer of products (protons and oxygen) from WOC (De Las Rivas &

Barber, 2004). Furthermore, the severe energetic and mechanistic

limitations under which photosynthetic water oxidation takes place require

efficient protection from toxic, partly oxidized intermediates that is ensured

by the high turnover rate of the PSII complex. Using the non-detergent

fractionation, a number of PSII complexes with different integrity were

isolated from thylakoid membranes (Veerman et al, 2007), each of them

representing different steps in the PSII repair cycle. Thus, PSII in vivo is a

highly dynamic system (for a recent review see (Suorsa & Aro, 2007) and

(Barber, 2002).

2.3. Mechanism of photosynthetic water oxidation

A crystallographic study by Ferreira et al. provided, for the first time, the

structure of the manganese cluster in PSII - an asymmetric cube of metal

ions, including three manganese atoms and a calcium that is linked to a

fourth manganese ion located at a longer distance from the cubic motif

(Mn4Ca cluster) (Ferreira et al, 2004). However, the high-energy X-rays and

long time exposure used in crystallography induced damage to the Mn4Ca

cluster due to reduction of manganese (Yano et al, 2005). The most reliable

up today structure of the Mn4Ca cluster in the WOC was obtained by

polarized EXAFS on PSII crystals (Yano et al, 2006) (Figure 3) and provided

more precise information about distances of the Mn-Mn and Mn-Ca bonds,

their bridging interaction, and oxidation levels.

The amino acid residues that provide ligands to the metal ions of the Mn4Ca

cluster belong mostly to the PsbA protein with one residue in the PsbC

protein: Asp170, Glu189, Glu333, His332, Asp342 and Ala344 residues of

PsbA as well as Glu354 of the PsbC protein. The question whether each

amino acid is bridging one atom in the Mn4Ca cluster (bidentate ligand) or

two metal ions remains open. Although so far the structure of Mn4Ca

complex has been resolved for only one organism, protein alignment

16

indicates that the amino acid sequence is fully conserved. Furthermore, the

Mn4Ca cluster is operating under severe thermodynamic restrictions of water

splitting, implying that there is no variation in structure of the Mn4Ca cluster

and that its chemistry is tightly tuned by the protein environment.

Figure 3. Model of the Mn4Ca cluster derived by polarized Mn EXAFS into the putative ligands obtained from the X-ray crystal structure. The spheres represent Mn (red), Ca (green), and the bridging oxygen ligand atoms (gray) (Yano et al.(Yano et al, 2006)).

In 1970, Bessel Kok and co-workers (Kok et al, 1970) developed a model

that is still relevant to explain the oscillation pattern of oxygen release under

exposure to short flashes. The Kok-cycle invokes a sequence of four photo-

oxidation steps of the Mn4Ca cluster (S-states) starting from S1 in dark-

adapted PSII. When, after the third flash, the Mn4Ca center achieves its

highest oxidation state, S4, it spontaneously returns to the lowest oxidation

stat, S0, due to the splitting of two water molecules accompanied by

production of O2 at the catalytic site (a process which is mirrored by the

reduction of the Mn4Ca cluster). In contrast to the O2, proton production is

distributed over the oxidation steps, following a pattern of 1:0:1:2

(Schlodder & Witt, 1999). The protons release during the terminal transition

occurs with a characteristic half-time of about 1.2 - 1.4 ms (Haumann &

Junge, 1994), which is observed also for both the reduction of the Mn4Ca

17

cluster and the release of O2. The O2 is produced and released only when,

upon illumination of PSII in the S3 state, the centre has reached it’s forth

oxidation state, S4. This third flash induces a cascade of chemical reactions,

discussed in details in (Rappaport & Diner, 2007) .

In a series of experiments employing time-resolved inlet mass spectrometry

(MIMS) and using labeled water H2O18, it was shown that the substrate

water in the WOC is exchangeable with bulk water in all S states and that

two substrate water molecules are characterized by different time constants.

It was concluded that for two substrate water molecules two binding sites

with different exchange kinetics are present in the WOC (Clausen & Junge,

2004; Hillier & Wydrzynski, 2004). Studies with elevated O2 pressures that

increased the concentration of the water splitting product (O2) were

conducted recently (Clausen & Junge, 2004). Under these conditions, the

reaction equilibrium of water oxidation was shifted toward its substrate and

a peroxide intermediate of water oxidation was detected.

The identity of the proton acceptor in the water splitting reaction has been a

matter of debate for decades (Junge et al, 2002). Limited methodological

approaches that allow monitoring of the proton transfer, in comparison with

electron transfer, led to ambiguity on this issue. The efficient removal of

protons from the WOC is crucial for the energetics of water splitting. Proton

dilution can provide an entropy gain of about 200 meV to the driving force of

the water oxidation reaction (Krishtalik, 1990). On the other hand, an

uncompensated positive charge accumulated on the Mn4Ca cluster would

generate an energetic barrier to subsequent oxidation steps (Dau &

Haumann, 2005).

The hypothesis that YZ, hydrogen bonded to His190 (Mamedov et al, 1998)

of the PsbA subunit, is directly involved in the catalytic water oxidation was

presented by Babcock and coworkers (Tommos & Babcock, 2000). It implies

removal of electrons from water, accompanied by removal of protons (the

hydrogen atom abstraction mechanism). This was the favored mechanism

for several years, however, it is now rejected based on the fact that the

18

distance between YZ and Mn4Ca in the PSII crystal structure is too long for

hydrogen atom transfer (Ferreira et al, 2004). Furthermore, the hydrophilic

channel in the PSII structure, which is thought to be responsible for the

proton transport from the Mn4Ca cluster to the lumen, is located too far from

the YZ residue (Ho & Styring, 2007; Murray & Barber, 2007).

The pKa of a primary proton acceptor should be sufficiently high to abstract

protons from the substrate water molecule. At the same time it should be

easily deprotonated and pass protons further along the proton transfer

pathway. McEvoy and Brudvig (McEvoy & Brudvig, 2004) proposed that

Arg357 of the PsbC protein is such a proton acceptor group. A recent paper

on a Arg357 mutant fully confirms the importance of this residue for the

water splitting reaction (Hwang et al, 2007).

There are several proposed mechanisms for the water oxidation reaction in

the PSII. In general, omitting details, they could be divided into three

hypotheses: i) a mechanism, that utilizes Ca2+ as a Lewis acid to

deprotonate a bound H2O; the resulting bound OH- creates a nucleophile

attack on O = Mn(V) bond that leads to O-O formation and reduction of the

Mn4Ca cluster; ii) a mechanism that allows the formation of a peroxide

intermediate bound to the Mn4Ca; and iii) accumulation of four positive

holes and four bases followed finally by an electrostatic repulsion of the last

proton leading to water splitting. The later mechanism assumes that

substrate water molecules remain intact (not oxidized, neither deprotonated)

until the terminal step of the reaction. For a recent review see (Renger &

Kuhn, 2007; Dau & Haumann, 2007 and Brudvig, 2007).

2.4. Role of calcium in PSII

The requirement of Ca2+ for the photoactivation of manganese-depleted PSII

(Chen et al, 1995) and vice versa, requirement of Mn2+ for the efficient Ca2+

binding (Adelroth et al, 1995), indicate a direct interaction between these

19

two metals in the WOC. The addition of Ca2+ decreased hydroxylamine-

induced reduction of manganese in PSII preparations and delayed the

subsequent release of Mn2+ in bulk (Mei & Yocum, 1991). There are at least

two binding sites for the calcium in the PSII, with different affinity depending

on the PSII isolation protocol and Ca2+ removing procedures used. 45Ca2+

experiments (Adelroth et al, 1995) show two dissociation constants for low

and high affinity calcium binding sites with KD of 25 - 60 mM and 0.5 - 1.7

mM, respectively. Among other divalent cations, only Sr2+ can replace Ca2+

at its binding site and restore O2 evolution (Boussac et al, 2004);

(Ghanotakis et al, 1984). This correlates with the acidity of divalent cations:

both Sr2+ and Ca2+ have higher acidities than other metals with similar

radius that bind to PSII (for a recent review see(Miqyass et al, 2007).

Several experimental approaches as well as computational models indicate

that the function of calcium in water splitting is to bind substrate water

molecule(s) in the Mn4Ca cluster (Siegbahn, 2002).

2.5. Role of bicarbonate in Photosystem II

In addition to the involvement of inorganic carbon (Ci) in the dark reactions

of photosynthesis (as a substrate for CO2 fixation in the Calvin cycle), it is

also required for optimal activity of PSII. Fifty years ago Warburg and

Krippahl (Warburg & Krippahl, 1958) demonstrated that CO2 enhances O2

production in isolated thylakoids. Their postulated role of bicarbonate (BC)

as the electron donor in PSII has been ruled out by recent measurements

using cyanobacterial and higher plant PSII samples. These findings

suggested that BC is a structural component of the PSII acceptor side where

it binds to a non-heme iron between QA and QB (Blubaugh & Govindjee,

1988). This conclusion was supported by X-Ray diffraction data, and the BC

molecule was included in the PSII crystal structure (Ferreira et al, 2004).

The possible role of BC on the donor side of PSII, however, remains

controversial. It has been shown that BC protects PSII from photo- and

thermo-inactivation as well as from over-reduction of the Mn

B

4Ca cluster by

20

hydroxylamine (Klimov & Baranov, 2001). Most of the experimental

evidence available for the donor-side BC effects came from reconstitution

experiments, where, after removal of Mn4Ca cluster, it was built up again

during photoactivation in the presence of Mn and Ca ions and BC

(Baranov et al, 2000). Electrochemical titration experiments led to the

conclusion that the higher capability of Mn-BC complexes to reconstitute O

2+ 2+

2

evolving activity in the PSII preparations lacking manganese is due to its low

oxidation potential of these complexes compared to hydrated Mn ion

(Dasgupta et al, 2006). The fact that BC accelerates the photoassembly of

the Mn

2+

4Ca cluster leaves open the possibility of its structural role in the

electron donor side of PSII.

An in vivo requirement of BC at the donor site of PSII was shown in the

cyanobacterium Arthrospira maxima (Carrieri et al, 2007). This strain is

growing at 0.2-1 M BC, pH 10; however, the intracellular concentration of

BC is strongly dependent on a Na+/BC transporter. A. maxima cells

resuspended in low BC media show a 30-50% decrease of PSII donor site

activity. The R357 residue of the PsbC protein was suggested as both a

potential BC binding site at the donor side of PSII (Ananyev et al, 2005) and

as a strong base that facilitates deprotonation of the substrate water

molecule bound to the Mn4Ca cluster at the beginning of a proton removing

pathway (McEvoy & Brudvig, 2004). The mutation of R357 residue led to

severely impaired water oxidation (Hwang et al, 2007). The pattern of O2

evolution per flash in this mutant is characterized by a high miss parameter

due to a defect in higher red-ox S-states.

A different role for BC in O2 evolution was proposed by (Metzner et al,

1979). He suggested that BC acts as a “shuttle” to bring solvent water to the

catalytic site. Thus, in this case, BC is the immediate precursor to O2 while

water is the ultimate source. Crucial for this hypothesis would be the

presence of a PSII-associated carbonic anhydrase (CA), to convert catalytic

amounts of CO2 to BC before it is lost to the atmosphere.

21

3. The Cah3 protein

CO2 in solution exists in equilibrium with BC and gaseous CO2 in the

atmosphere. CO2 can freely penetrate biological membranes, while BC can

not because it is not soluble in lipids. The interconversion of BC with CO2

proceeds slowly at physiological pH, therefore, living organisms evolved a

specific enzyme to speed up this process – CA. The CA was isolated for the

first time in 1933 (Meldrum & Roughton, 1933) and since then this key

enzyme has been intensively studied.

Figure 4. Ribbon diagram of the Cah3 protein with secondary structure elements labelled. Helices are shown in red, ß-strands in yellow, and cysteine residues are indicated in blue. The magenta sphere denotes the location of the Zn2+ ion. (Swiss Model)

There are three evolutionarily unrelated families of CA, α−, β− and γ− carbonic

anhydrases that do not have any significant homology in their sequences

(HewettEmmett & Tashian, 1996). All of them contain Zn2+ at the active site

and exhibit similar mechanism of catalysis. αCA is a single protein of ~29

kDa carrying a Zn2+ ion bridged by histidine residues which are fully

conserved for this CA family. αCA is characterized by a high turnover rate of

about 100 reactions per second (Lindskog, 1997). In the metabolism of

algae and plants, the CA family serves different functions. CA plays its

22

primary role in carboxylation/decarboxylation reactions, and therefore it

functions in algal Carbon Concentrating Mechanisms (CCM), in ion transport,

in pH homeostasis and in the production of carbon skeletons in

mitochondria.

The first algal intracellular αCA (Cah3) was identified in the green algae

Chlamydomonas reinhardtii and was shown to be localized in the thylakoid

lumen (Karlsson et al, 1998). Later, immunologold analysis supported that

Cah3 is situated on the lumenal side of thylakoid membranes. Evidence for

CA activity in the thylakoid fractions of other green algae (Tetraedron

minimum and Chlamydomonas noctigama) has been documented (van

Hunnik et al, 2001). The PSII-associated CA activity has also been identified

in higher plants. However, the source of this activity remains enigmatic. The

mechanism for CO2 hydration of the human αCA, CAII isozyme, is well-

established. This mechanism utilizes Zn2+ to act as a Lewis acid to stabilize a

nucleophilic OH- (lowering the pKa of H2O by up to eight pH units). The rate-

limiting step for the overall reaction is an intramolecular proton transfer

from zinc-bound water via fully conserved His64 ultimately to the bulk

solvent. The essential elements of this mechanism are depicted in the

following scheme:

H2O HCO3-

E-Zn2+—OH-+CO2 E-Zn2+—HCO3

- E-Zn2+—H2O E-Zn2+—OH-+H+ i) the nucleophilic attack of the Zn-activated OH- on CO2

ii) the exchange of the resulting HCO3- for a water molecule

iii) regeneration of the Zn2+ - OH- form of the enzyme

4. The PsbO protein

The extrinsic PsbO protein has been intensively studied since its discovery

almost 30 years ago (Kuwabara & Murata, 1979). The X-ray crystal structure

23

of a cyanobacterial PSII provides for the first time a detailed structure of

PsbO bound to the PSII (Ferreira et al, 2004). The PsbO is orientated at

about 40o angle relative to the membrane plane with the C- and N-termini

facing towards the lumen (see Figures 1, 5). The PsbO protein is a cylinder

with a length of 35 Å and a diameter of 15 Å. Its folding represents a β-

barrel composed of eight antiparallel β-strands with hydrophobic amino acid

residues exposed towards the inside. In addition, the PsbO has random coils

and turns, inserted between the β-strands, together with a short distinct α-

helix. The β barrel folding is known as a stable structure in many proteins,

for example in aquaporins (Cheng et al, 1997). In contrast, the hydrophilic

loops are stabilized by interaction of the PsbO with other PSII proteins and

are more flexible in the absence of those interactions, i.e. in solution. An

NMR study on overexpressed isolated PsbO corroborates that this protein

has two dynamically defined parts. About 40% of the protein has a high

flexibility on the ps to ns time scale and another part is rigid, implying that

PsbO consists of a stable hydrophobic core together with highly flexible

domains (Nowaczyk et al, 2004). The PsbO protein has a fully conserved

disulfide bridge between the N-terminal loop and the β1 strand (Cys19 and

Cys44 in T. elongatus, corresponding to Cys28 and Cys51 in the Spinacia

oleracea protein sequence). PsbO is attached to the lumenal side of the

thylakoid membrane via electrostatic interactions with extrinsic loops of the

PSII proteins: PsbA, PsbB, PsbC, and PsbD (De Las Rivas & Barber, 2004).

The cyanobacterium lacking PsbO protein, ΔpsbO mutant, exhibits

photoautotrophic growth and almost normal levels of PSII reaction centres

(Burnap et al, 1992). At the same time ΔpsbO mutants revealed modified Yz

reduction kinetics and a decline in photoactivation of the PSII (Burnap et al,

1996). The increase of the quantum yield of photoactivation is consistent

with the hypothesis that PsbO controls accessibility and stability of the

Mn4Ca cluster in the WOC (Burnap et al, 1996; Mayes et al, 1991). Since

biochemical and molecular genetic studies have indicated that MSP and cyt

c-550 each appear to stabilize the WOC, a possible interaction between the

24

two proteins was investigated by deletion analyses. The experiments

demonstrated that the single-gene deletion mutants of the psbO and psbV

genes (ΔpsbO and ΔpsbV, respectively) are able to grow autotrophically, in

contrast to the double-deletion mutant ΔpsbO: ΔpsbV (Shen et al, 1995).

In eukaryotic PSII the situation is quite different. A C. reinhardtii mutant

lacking PsbO is not able to assemble the PSII reaction centres and grow

photoautotrophically (Mayfield et al, 1987). A recent study using RNAi to

suppress both psbO1 and psbO2 genes in Arabidopsis thaliana produced

similar results (Murakami et al, 2005). Additionally, the limited proteolysis of

PsbO isolated from various organisms results in different degradation

products (Tohri et al, 2002). There are differences in the binding of extrinsic

proteins to PSII between higher plant and algae that has been brought

forward by cross-reconstitution experiments (Enami et al, 2000). In plants,

PsbO provides binding sites for attachment of PsbP and PsbQ, probably by

electrostatic interactions or salt bridges. It is notable that under

physiological conditions a significant pool of extrinsic PSII proteins is

unassembled to PSII and remains free in lumen (Hashimoto et al, 1996).

This pool is essential for the reassembly of PSII during the repair cycle.

Moreover, the comparison of photoinhibition efficiency in the presence and in

the absence of the PsbO protein under otherwise similar conditions clearly

demonstrates that it prevents PsbA from aggregation and thereby facilitates

its turnover in the PSII repair cycle (Komayama et al, 2007).

Extraction of PsbO protein from PSII particles by salt washing inhibits normal

oxygen evolution so that it can only be detected at non-physiological high

concentrations of CaCl2 (Bricker, 1992) (for a review see (De Las Rivas &

Barber, 2004). At lower Cl- concentrations manganese atoms are released

from the WOC upon dark incubation (Ono, 1983). Removal of the PsbO also

affects the turnover and stability of the higher redox S states of the WOC

(Vass et al, 1987). Moreover, O2 evolution in PSII lacking the PsbO protein is

susceptible to donor side photoinhibition (Bricker, 1992). Thus the PsbO

25

protein is an important constituent of a fully intact WOC and essential for the

stabilization of a functional Mn4Ca cluster. The MIMS experiments using

labeled water H2O18 demonstrate that in the absence of PsbO the rate of

substrate water exchange between the WOC and bulk was slowed down

(Hillier et al, 2001). Thus, the possible function of the PsbO protein in the

WOC could be stabilization of Mn4Ca cluster and regulation of substrate

water supply.

Figure 5. Ribbon diagram of the PsbO protein, with secondary structure elements labelled. Helices are shown in red, ß-strands in yellow, and cysteine residues are indicated in blue.

Metal ions affect the structure of PsbO in vitro. Addition of Ca2+ induces the

decrease of the unordered structure and β-sheet, and a substantial increase

of α-helix, as was shown by FTIR spectroscopy. In contrast, other

experiments demonstrated that the β-structure is replaced by random coil,

or that it is no significant Ca2+ -induced changes of the secondary structure.

The discrepancy between results obtained in different in vitro experiments

for higher plant PsbO as well as between higher plant and cyanobacterial

PsbO can be due to different experimental conditions used (pH and/or

different amount of CaCl2 was present, depending on the isolation method).

Furthermore, there is no known Ca-binding motif in the PsbO sequence. The

helix-loop-helix folding typical of “E-F hand” of Ca2+-binding motif proteins is

missing in the PsbO tertiary structure (Ferreira et al, 2004). Instead, the

26

hemolysin-type Ca2+-binding by parallel β-roll structures may have

similarities to the PsbO tertiary structure (Heredia & De Las Rivas, 2003). In

a recent study, the Ca2+ ion bound to the PsbO was identified using the

anomalous diffraction data at 5.5 keV of the PSII crystals from T. elongatus

(Murray & Barber, 2006). The authors show that Ca2+ is ligated by E54,

E114 and H231 residues in the PsbO protein. However, among these

residues only E114 is conserved in higher plants and green algae. It is

important to note also that PSII core complexes used in this study were

isolated and kept in the presence of 10 mM CaCl2.

5. Red-ox regulation in lumen

The electrochemical gradient generated across the thylakoid membrane, as

well as reactive oxygen species accumulated as a side product during the

photosynthetic reactions, cause light-induced shifts in the red-ox potential of

the lumen in light. The ferredoxin-thioredoxin system of chloroplasts,

responsible for the activation of stromal enzymes in the light by reduction of

S-S bonds has been well established. The red-ox state of plant thioredoxins

directly depends on the electron flow in the thylakoid membrane, because

they are reduced by the final electron acceptor in the chain, ferredoxin or by

the NADPH-dependent thioredoxin reductase. Glutaredoxins are also

components of redox regulation in chloroplast (Michelet et al, 2006). They

derive their function by incorporating glutathione, a trimeric peptide, into

the target enzymes via thiol groups.

Recent results indicate that there are also redox-regulated enzymes in the

thylakoid lumen. The activity of a lumenal immunophillin, FKBP13, is

reversibly inhibited by reduction of the disulfides (Marcaida et al, 2006).

However, the system that catalyzes this red-ox regulation in the lumen is

still unknown. The hypothesis of redox-regulation in the whole chloroplast

has been recently proposed (Buchanan & Luan, 2005). Lumenal enzymes are

27

activated in light by oxidation of SH groups, in contrast to the stroma

enzymes, where, reduction of disulfides leads to an increase of activity.

When all chloroplast proteins were analyzed with respect to potential

thioredoxin targets, the PsbO was identified, together with other well-

established redox-regulated proteins of the stroma (Lemaire et al, 2004).

The importance of the S-S bridge for the PsbO structure and function

remains controversial. Tanaka and Wada for the first time demonstrated that

PsbO contains S-S bond and that the reduced protein is not folded and can

not restore O2 evolution (Tanaka et al, 1989). The same conclusion came

from Cys20S substitution in PsbO from Synechocystis (Burnap et al, 1994).

However, later it was reported that the mutation removing the S-S bond

does not affect the function of the PsbO since the mutated protein was able

to bind to PSII and restore O2 evolution (Betts et al, 1996; Wyman & Yocum,

2005).

II. Results

1. The lumenal carbonic anhydrase, Cah3, is required for functioning

and stability of the electron donor side of PSII

To elucidate the role of the lumenal CA in PSII, first of all we have

characterized features of PSII from the Cah3-less mutant of Chlamy, cia3

(Paper I). To compare the efficiency of light energy conversion in wt and

cia3 mutant cells, we measured the light response curves of O2 evolution.

Under standard growth conditions, mutant and wt cells have similar

efficiency of light utilization as well as similar electron transport rate in both

thylakoids and PSII preparations. When the sensitivity to high light

treatment was compared, the difference between wt and cia3 cells was

revealed. The rate of photosynthesis in wt cells was reduced by

approximately 35% of control values vs. 80% in the cia3 mutant. When wt

thylakoid membranes were photoinhibited in the presence of EZ, a specific

inhibitor of CA activity, the rate of PSII electron transport identical to the

28

cia3 mutant was obtained. At the same time, EZ did not cause an effect on

wt thylakoid membranes without exposure to high light, as well as on

thylakoids from the mutant cia3.

In order to understand whether the photoinduced damage in the cia3 mutant

occurs on the donor or acceptor side of PSII, wt and cia3 mutant thylakoids

were subjected to light treatment in the presence of DCBQ, which accepts

electrons from the QA and, therefore decreases acceptor side photoinhibition.

The addition of DCBQ completely abolished the photoinduced damage of the

wt PSII, indicating that it occurred at the acceptor side of PSII. On the other

hand, DCBQ was not able to prevent photoinhibition in cia3 thylakoids

because in the mutant the donor-side photoinhibition takes place. Indeed,

the addition of BC during the high light treatment to cia3 mutant thylakoids

protected them from damage. Thus, in the absence of an active CA, the

donor side of PSII is more sensitive to light excess, which could be

compensated by the addition of BC.

Moreover, the addition of BC to non-photoinhibited cia3 mutant thylakoid or

BBY preparations stimulated O2 evolution rates to ~40% higher than in

controls. The BC effect was increased upon lowering the pH values from 7.6

to 5.5. In wt preparations, no stimulation of O2 evolution was found. The

higher PSII electron transport rates measured in mutant thylakoids in the

presence of BC can be explained if the mutant contains a higher number of

PSII reaction centres. We estimated the concentrations of PSII and PSI by

measuring the photoinduced absorbance changes related to the

photoreduction of pheophytin and photo-oxidation of P700, respectively. Our

data indicate that the ratio of PSII/PSI is increased in the mutant cells (2.44

0.1 vs. 1.58 0.2 in wt cells). Measurements of light-induced electron

transfer to the electron acceptor DCPIP – Hill reaction - confirmed these

observations. Therefore, we conclude that the cia3 mutant cells compensate

for their less efficient PSII donor side by overproducing PSII reaction centres

in order to maintain an optimal electron transport.

29

In a next step of our research, we have improved our experimental system.

In addition to intact cells, thylakoids and isolated PSII membrane fragments

from either wt or cia3 mutant, the PSII activity was measured in the

presence of overexpressed and purified Cah3 protein together with its

substrate, BC (Paper II). All measurements were conducted in a buffer

initially free of Ci at pH 5.5, where highest stimulation of O2 evolution was

observed (see above). This value is close to the pH of the thylakoid lumen in

the light, and at this pH, most of the Ci exists as dissolved CO2.

In the presence of Cah3 protein, the BC-induced increase in O2 evolution in

cia3 mutant PSII membrane fragments was as high as ~60-80 % and the O2

evolution rate reached the wt rate if calculated per reaction centre. BC alone

was unable to restore O2 evolution fully. Moreover, this stimulatory effect by

Cah3 and BC added together was achieved at 10-fold lower BC concentration

than without enzyme. Addition of EZ together with Cah3 prevented the

reactivation of O2 evolution. Contrary to Cah3, the bovine carbonic

anhydrase, which belongs to the same CA family as Cah3, did not stimulate

O2 evolution despite its high activity. Thus, Cah3 specific enzymatic activity

is required to restore O2 evolution in the mutant PSII.

Because BC has been shown to have acceptor side effect in PSII (Blubaugh &

Govindjee, 1988), it was of crucial importance to measure directly whether

the stimulatory effect of BC was caused by a donor side effect or not. To

address this question, we measured flash-induced changes of prompt Chl

fluorescence, which reflects the reoxidation of reduced QA by QB(Dau, 1994).

There was no difference in the decay kinetics in the absence and in the

presence of Cah3 or BC, strongly suggesting that the Cah3/BC effect is not

associated with changes at the acceptor side of PSII.

B

The next step in the understanding of the features of the BC effect in the

PSII was the flash-illumination experiments. The rate of O2 evolution under

continuous light depends on both the number of active centers, and the

30

rate-limiting step in each center. To discriminate between these two

possibilities, we measured the kinetics of PSII reactions in the elementary

act, i.e. under excitation with single-turnover flashes. The amplitude of the

flash-induced O2 release was not changed in the presence and in the

absence of Cah3/BC, implying that the number of active centres remained

unaffected. At the same time, the rise of O2 in mutant PSII was accelerated

by the addition of Cah3 and/or BC. In the wt PSII, on the other hand, there

was no effect of these additions (for a more detailed analysis see Paper II).

2. Cah3 and the proton removing from the WOC

What is then the limiting factor for the O2 evolution rate in the mutant PSII?

Our hypothesis is that Cah3 stimulates O2 evolution via its substrate/product

BC as a carrier facilitating proton removal from the vicinity of the WOC. To

test this hypothesis, we used neutral red (NR), an amphiphilic buffer known

to bind protons on the surface of thylakoid membranes (Auslander & Junge,

1975). The stimulatory effect of NR on the rate of O2 evolution was as great

as for the joint addition of BC and Cah3, while there was no effect observed

in wt PSII. While NR effectively restores O2 evolution in mutant PSII, other

buffers with suitable pKa do not. As opposed to these buffers, NR is

amphiphilic and is aligning along the membrane surface where it accepts

protons directly from the donor(s) instead of accepting protons from the

lumen bulk. The fact that NR mimics the effect of BC and Cah3 supports the

notion that Cah3 accelerates O2 evolution via protons removal from the

WOC.

A strong support for a proton limitation in mutant PSII comes from the

experiments when exchangeable protons were replaced by deuterons. The

kinetic isotope effect of the O2 evolution in the mutant PSII was 1.78. To

best of our knowledge, such a significant kinetic isotope effect on O2

evolution was not reported before. In contrast, in the wt PSII the

replacement of H2O by D2O causes only minor changes (~15%). The kinetics

of electron transfer is virtually the same in H2O and D2O (Christen & Renger,

31

1999). In marked contrast, the proton release from the WOC, especially in

the terminal step of the water oxidation reaction, exhibits greater isotope

effects (Haumann et al, 1997;,Karge et al, 1997). The result obtained for

the mutant PSII in a D2O-based solution revealed a kinetic solvent isotope

effect consistent with proton transfer being the rate-limiting step of the

oxygen evolution in the absence of Cah3 activity and BC.

Further support for a role of the Cah3/BC system in proton removal from the

Mn complex comes from our time-resolved measurements of flash induced

delayed chlorophyll fluorescence (DF). In the S2→S3 and S3→S4→S0

transitions of the WOC, an essential proton-release step has been suggested

to precede the electron transfer (Dau & Haumann, 2007). In the time

course of DF after the third flash, the S3→S4 transition is reflected in the

decay from the initial value to the plateau reached at about 1 ms. The

subsequent DF decay in the millisecond time domain reflects the reduction of

the YZ•+ radical and the simultaneous dioxygen formation (Zankel, 1971). In

the mutant PSII, the rate of proton removal in the S4-formation step was

reproducibly accelerated by about 25% by the addition of BC, but not in the

wt PSII (for the more detailed analysis see Paper II). The DF decay was also

accelerated by addition of BC in the S2→S3 transition, but not in S1→S2

transition, which does not involve any proton removal from the Mn4Ca

cluster. Indeed, the DF decays measurements suggest that the BC addition

is required for restoration of the proton-removal rate in the mutant PSII and

thereby support the suggested role of BC as a proton acceptor and/or

carrier.

3. Cah3 protein is associated with the PSII

For the above-mentioned hypothesis to be correct, it requires that the

thylakoid CA is closely associated with PSII. In fact, an enrichment of Cah3

in PSII preparations was obtained, compared with isolated thylakoid

membranes and total cell extracts (Paper I). The Cah3 protein was not

detectable in the PSI fraction. Furthermore, the leader sequence of Cah3,

32

typical for lumen-targeted proteins (Karlsson et al, 1998) together with

immunolocalization of Cah3 on the lumenal side of thylakoid membranes

(Mitra et al, 2005) strongly suggest that Cah3 is located at the donor side of

PSII. Both sequence analysis and the experimental data on removal of Cah3

by salt treatment predict that Cah3 is not a membrane-spanning

polypeptide, but rather is attached extrinsically by ionic and/or hydrophobic

interactions.

Following reactivation of the O2-evolving activity of mutant PSII by

overexpressed and purified Cah3, the samples were separated into

membrane-bound and soluble fractions and then were subjected to western

blot analysis (Paper II). All Cah3 protein was detected in the pellet,

indicating that the Cah3 protein binds to PSII. The specific association is

proven by the low KD of about 0.1 μM. In agreement with the rebinding

experiments, the greatest stimulation of O2 evolution by Cah3 was observed

at a 1:1 protein to PSII reaction centre ratio. It seems therefore as if

stimulation of O2 evolution is very precisely regulated by the stoichiometric

binding of the Cah3 protein to the PSII docking site.

4. Relevance to the PSII from the other organisms

So far C. reinhardtii is the only organism with a characterized CA associated

to PSII. In the cyanobacteria no CA activity associated to PSII has been

detected (Hillier et al, 2006). While BC is also clearly required at the donor

side of cyanobacterial PSII (Carrieri et al, 2007), the BC concentration is

kept by active BC transporter. In PSII preparations from higher plants, CA

activity has been found (Hillier et al, 2006; Moubarak-Milad & Stemler,

1994) although no PSII-associated enzyme has yet been characterized. A

database search of higher plant genomes reveals 35 candidates with

putative location in the chloroplasts. The thylakoid membrane proteome of

A. thaliana chloroplasts also revealed an αCA homologe (Friso et al, 2004).

The reactions at the PSII donor side have been inferred to be almost

invariant to the evolution from cyanobacteria to higher plants (Karge et al,

33

1997). It has been reported that the O2 release step may be critical for the

energetics of photosynthetic water oxidation (Clausen & Junge, 2004). Our

findings suggest that also proton removal from the catalytic Mn4Ca complex

of PSII is also a process that can potentially limit photosynthetic dioxygen

formation, at least at the absence of available BC and for saturating

illumination at the lumenal ‘working pH’ of about 5.5. Our data support the

crucial role of proton-removal steps for energetics and mechanism of

photosynthetic water oxidation (Krishtalik, 1986; Dau & Haumann, 2007)

and shift the focus in PSII research from the electron transfer reactions

towards the role of the associated proton movements.

5. PsbO and the proton removing from the WOC

Another potential candidate to be involved in proton transfer from the

Mn4Ca cluster is the PsbO protein (see Introduction part). As was found

earlier (Oda et al, 1994), ~12 carboxylic acid residues of the PsbO protein

from spinach have a shifted pKa to of 5.7 (pKa in water around 4.0). A

similar shift in pKa of carboxylic acid residues due to a short distance

between them has also been shown for some other proteins: ribonuclease

H1 (Chen et al, 2000), CDd2 protein (McIntosh et al, 1996), xylanase

(McIntosh et al, 1996). According to our hypothesis (proposed in the Paper

III) carboxylic groups with high pKa in the PsbO protein form a specific

cluster, of carboxylic groups (CCG). Based on PSII 3D structure (Pdb entry

1S5L), we identified 16 glutamic and aspartic acid side chains forming the

CCG, including seven fully conserved residues and five residues conserved

among higher plants or cyanobacteria only. The average distance between

the neighbouring residues in the cluster was estimated to be 7.5 Å. Similar

distance between two or more acidic groups was found to be responsible for

a common negative field and high efficiency of proton transfer in

bacteriorhodopsin and in proton transporters with known 3D structure

(Mulkidjanian et al., 2006).

34

As was discussed in the introduction, dilution of protons can provide the

necessary entropy gain to the driving force of water splitting. In contrast to

previous models, we propose that such effect could result not from the

dilution of protons into the bulk via proton channel (De Las Rivas & Barber,

2004; Ishikita et al, 2006), but can be achieved by a proton accepting

antenna of the CCG. Importantly, the charge delocalization of the CCG will

ensure a readiness to promptly accept protons from the WOC. The proton

transport via hydrogen-bonded water molecules on protein surfaces implies

a Grotthuss mechanism, characterized by a cascade of proton jumps, so that

the proton arriving at the final acceptor is not the same as the one that

leave the source (Cantini et al, 2006; Sacher et al, 2005). This mechanism

requires a precise distance and orientation of the carboxylic side chains and

can conceivably be regulated by protein dynamics.

The function of the Mn4Ca cluster requires a rather hydrophobic

environment; therefore the accessibility of substrate water might be a

problem. The CCG is located in a hydrophilic region of the PsbO protein.

Nevertheless the K159-K186 region is shielded from cross-linking agent

when the PsbO protein is associated with PSII (Frankel & Bricker, 1995).

This, as well as analysis of the 3 D-structure of PSII suggest that the CCG

are located in a hydrophilic pocket and could bind a local pool of bound

substrate water. This would fit the data demonstrating that in the absence of

PsbO the rate of substrate water exchange is slowed down (Hillier et al,

2001). Thus, we propose that a cluster of conserved glutamic and aspartic

acid residues with an optimal pKa, considering the lumenal pH, in the light

acts as a buffering network in the PsbO providing efficient acceptors of

protons derived from water splitting. Our hypothesis predicts that protons

generated at the WOC are not released into the lumen via a specific channel,

as generally thought (De Las Rivas & Barber, 2004). Instead, a localized,

efficient proton transfer conduit (antennae) on the lumenal side of the

thylakoid membrane is responsible for removing protons from the WOC and

to the lumen bulk.

35

6. Structural dynamics of the PsbO is necessary for its function,

possible GTP binding

The characteristic of PsbO protein in solution is a matter of considerable

debate. It was even suggested that the protein has no compact structure

and therefore could be classified as a natively unfolded protein. According to

our results (Paper IV), the urea-induced unfolding/refolding of the PsbO is

characterized by the high degree of cooperativity and fits to a two-state

model with an average value of conformational stability of the dG(H2O) = 14

± 3 kJ/mol and the cooperativity of the transition m = 6.2 kJ/mol. These

parameters are quite similar to those of classical globular proteins of same

size with well-defined tertiary structure. Thus, our data provide

unambiguous evidence that the PsbO keeps its folding in solution.

Our hypothesis is that the PsbO protein undergoes pH induced

conformational changes in the physiological pH range (Paper V). ANS

fluorescence experiments suggest that the accessibility of the hydrophobic

regions of the PsbO increases upon the protonation (from pH 7.2 to pH 5.7).

In its acid conformation, the PsbO protein can bind both Ca2+ and Mn2+.

Instead of a decrease of metal binding upon lowering of pH due to the

competition with H+ for the negatively charged binding sites, as in well

studied protein, α lactalbumin for example, the PsbO interaction with metals

is much stronger at acidic pH. We suggest that this could be due to a re-

arrangement of the PsbO at low pH resulting in a greater accessibility of

negatively charged groups involved in Ca2+ and Mn2+ binding.

The additional Mn2+-induced increase of ANS fluorescence in the pH region

between 6.5 and 5.5 may reflect an opening of the hydrophobic core upon

Mn2+ binding. The same behaviour was reported for Ca2+-sensor proteins

involved in transduction of intracellular signals that expose a hydrophobic

area in response to Ca2+ binding. Binding of metals usually leads to

stabilization of protein structure, accompanied by the shift of the melting

curves to a higher temperature, which is not the case for the PsbO. Ca2+ and

36

especially Mn2+ (ΔT 20 oC) considerably lower the protein melting point at pH

5.7; this is accompanied by an increase of the cooperativity factor of the

thermal transition. The simultaneous lowering of melting point and the

increase of the transition cooperativity may reflect that Mn2+ binding induces

the formation of a local, well-structured, domain within the protein molecule.

The fact that the far-UV CD spectrum of the PsbO in the presence or absence

of metals is virtually the same indicates that the fraction of the secondary

structure elements, presumably β-strands, is unaffected.

All our results (thermal unfolding monitored by intrinsic fluorescence and

CD, fluorescence of hydrophobic probe ANS, determination of the metal

binding constants) are in agreement with the conclusion that the interaction

of the PsbO with Ca2+ and Mn2+ tacks place at pH 5.7 but not at pH 7.2. The

changes of pH in this range occur in the lumen depending on illumination.

Our model of pH dependent Ca2+ and Mn2+ binding to the PsbO is depicted in

Figure 8, Paper V. All conformational changes in this model are reversible.

The PsbO protein does not interact with Ca2+ and Mn2+ in darkness when the

lumenal pH is around pH 7.5. Light-induced protonation of the PsbO leads to

exposure of hydrophobic residues. This assumes that protons released

during water oxidation bind to the PsbO and change it from a closed to an

open conformation. Light conformation of the PsbO is able to bind both, Ca2+

and Mn2+. This binding, in turn, induces a conformational change leading to

the additional exposure of hydrophobic patches of the PsbO protein.

Therefore, our conclusion is that, the PsbO interacts with both Ca2+ and Mn2+

in pH dependent manner within the physiological pH range.

Light induced lowering of pH in the lumen might lead to conformational

changes of PsbO, so that its function, whatever it is, becomes impaired.

Several functional implications may be suggested: i) the high flexibility of

PsbO in the light is important for regulating accessibility of substrate water

to the WOC and removal of protons from the WOC, ii) the PsbO binds Ca2+

and Mn2+, released in PSII repair cycle, iii) the PsbO is involved in a

37

reversible dissociation of the inorganic core of the WOC under physiological

conditions.

In accordance with our hypothesis, the recent work reveals the importance

of protein–protein interactions within the PSII and suggests that

conformation, which increase flexibility of the PsbO, is essential to its

function the water splitting (Williamson et al, 2007). Other experiments have

also indicated that side chains of PS II proteins with pKa values around 5.7

(as a pKa of CCG) are important for water oxidation. The pattern of proton

release from the PS II core complexes can be satisfactory simulated by

deprotonation of acidic groups with a pKa of 5.7 (Schlodder & Witt, 1999),

supporting a direct participation of these residues in proton transfer.

Further, the flash induced FTIR difference spectrum gives direct evidence for

deprotonation of glutamic and aspartic acid residues of the PsbO with high

pKa during the S1 to S2 transition (Hutchison et al, 1999).

We have also tested whether the acid conformation of PsbO exists when it is

bound to the PS II under illumination by the study the gentle extraction of

PsbO from PSII in light and darkness. Our results demonstrate that

illumination weakened the interaction of the PsbO with PS II, and that there

is a positive correlation between the amount of extracted PsbO and release

of inorganic core of WOC: Ca2+ and Mn2+ ions. Pre-incubation of PSII in

darkness at high pH prevents PsbO to form the “open conformation” and

also abolishes the effect of illumination. However, we can not rule out the

possibility that the PsbO docking site is affected by illumination. More

experiments are required to solve this problem. Our results suggest that the

local pH effect caused by protons released from PSII may be more important

for PsbO conformation than that of the bulk buffer.

Not only pH and metals, but also GTP induces conformational changes in the

PsbO protein in solution, as was detected by circular dichroism and intrinsic

fluorescence spectroscopy (Paper VI). Isolated PsbO exhibits a low GTPase

38

activity, which is enhanced when the protein is associated with the PSII core

complex. GTP stimulates the dissociation of PsbO from PSII under light

conditions known also to release Ca2+ and Mn2+ ions from the oxygen-

evolving complex and to induce degradation of the PSII reaction centre D1

protein. We propose the PsbO-mediated GTPase activity associated with PSII

in higher plant, which has consequences for the PSII repear cycle.

7. Red-ox regulation of the PsbO and Cah3 proteins

The PsbO is the only known protein in higher plant PSII carrying a disulfide

bridge, therefore it is a potential target for redox-regulation in the thylakoid

lumen. We applied urea gradient electrophoresis to study unfolding and

refolding of spinach PsbO and to estimate the contribution of the S-S bridge

to its stability (Paper IV). The unfolding transition of the reduced PsbO is

shifted towards lower urea concentration by 2 moles, so that the reduced

PsbO is not folded even without uUrea. Such a pronounced shift of the

transition suggests that the single S-S bond has a serious impact on the

stability of the PsbO as a whole. The stabilization of proteins by disulfide

bridges, located in the interior of the protein core is well established. By

contrast, the single S-S bridge of the PsbO is a peripheral, attaching only the

N-terminal loop to the external surface β-barrel. On the other hand, the β-

barrel domain seems to be responsible for the stability of the PsbO. The

eight-stranded antiparallel β-barrel is stabilized by an extensive hydrogen

bond network linking all β-strands and the tightly packed hydrophobic core.

To address this question, we calculated the number of residue-residue

contacts in the PsbO, related to the stability of different parts of protein

molecules. This mapping identified two clusters with the highest contact

density in the PsbO and Cys51 is located in the centre of one of these

clusters. In general, any perturbation of the packing interactions in the

regions with high contact density leads to destabilization of a protein. We

propose that the reduction of the S-S bridge, which increasing the flexibility

39

of the N-terminal loop, perturbs the interactions in the highest contact

density cluster and therefore destabilize the protein. Interestingly, the single

Trp241 of higher plant PsbO protein is located in the centrum of highest

contact density cluster together with S-S bridge. The close contact between

Trp241 and S-S bridge explains the rare fluorescence of the PsbO protein

when Trp241 emission is strongly quenched by disulfide (Paper VII).

The Cah3 protein contains three Cys residues. Two of them are involved in

intramolecular S-S bridge, and the third possibly acting as a ‘redox-sensor’

(on-going project with the group of Prof. G. Wingsle and project student R.

Ågren). Reduction causes a loss of enzymatic activity of the Cah3 due to the

breaking of the intramolecular S-S bridge, which would impair structural

integrity of the protein. The time-dependent measurements after DTT

addition indicated possible two-phase reaction kinetics. The free Cys55

residue of Cah3 is not directly related to activity, but may indirectly regulate

it via dimerisation of the protein. Immunoblot analysis of over expressed

and isolated Cah3 protein treated with β-ME shows a single 28 kDa band. In

the absence of β-ME an additional broad band of 50-55 kDa is revealed,

containing dimers of the protein. Dimerisation takes place in isolated Cah3

as well as in PSII membrane fragments, and in both cases it increased in a

non-reducing environment. Therefore, the free Cys55 residue could regulate

Cah3 protein activity and/or location due to the dimers formation. The Cah3

protein was also subjected to treatment with GSH/H2O2 mixture, resulting in

GS(O)SG. This agent has been reported as a more potent glutathionylating

agent than GSSG (Tao & English, 2004). According to mass spec analysis,

m/z ratios shifted upwards of about 450 Da in treated sample indicating

that, probably, one glutathione has bound to the protein. Previously, a β-

type carbonic anhydrase from chloroplast stroma has been identified as a

glutaredoxin target (Rouhier et al, 2005), indicating that this group of

enzymes is redox regulated. Whether the α-type Cah3 protein in the

thylakoid lumen is targeted by red-ox regulation remains to be investigated.

8. A comparative analysis of two lumenal proteins: PsbO and Cah3.

A comparative analysis of PsbO and Cah3 based on literature results and

own data is presented in Table 1.

Table 1. Structural features of PsbO and Cah3 proteins

Properties

PsbO

Cah3

Encoded

Nucleus

Nucleus

Location

Attached to the lumenal site

of thylakoid membrane

Attached to the lumenal site

of thylakoid membrane

Metals content

Ca2+ and/or Mn2+

Zn2+

Interactions

PsbA, PsbB, PsbC, PsbD,

PsbP, PsbQ

unknown

Secondary structure

β

β

Stability

thermostable

regular

Redox-regulation

intermolecular S-S bridge,

active in oxidized form

intermolecular S-S bridge active in oxidized form,

dimerisation, gluthationation

PSII activity in the absence of protein

~20-25%

~50%

Requirement for the reconstitution of O2

evolving activity

2-10 mM CaCl2

1-2 mM KHCO3

Overexpression

pET32, (pET28)

pET32

40

III. Conclusions and outlook

The results presented in this thesis highlight the importance of the protein

matrix in the functioning of the water oxidation complex. Two lumenal

proteins associated with the thylakoid membrane have been studied, PsbO

and Cah3. Both of them are required for optimal function of the water

oxidation complex. We show for the first time that a lumenal carbonic

anhydrase, Cah3, regulates the water oxidation reaction. We propose that

Cah3 promotes the removal of protons from the water-oxidizing complex by

locally providing bicarbonate, which may function as a proton carrier. The

PsbO protein, an important constituent of the water-oxidizing complex, is

the only extrinsic protein conserved in all known genomes of oxygenic

phototrophs (De Las Rivas et al, 2004). Despite extensive studies, however,

its function is still unknown. Our results led us to the conclusion that the

PsbO protein undergoes a pH dependent conformational change that

influences its capacity to bind calcium and manganese. We further propose

that light-induced structural dynamics of the PsbO is of functional relevance

for the regulation of proton release and for forming a proton sensing -

proton transporting pathway. The cluster of conserved glutamic and aspartic

acid residues in the PsbO protein acts as a buffering network providing

efficient acceptors of protons derived from substrate water molecules. Both

Cah3 and PsbO have a conserved disulfide bridge and are red-ox regulated,

at least in vitro.

Further research will be focused on:

• The role of red-ox regulation of Cah3 and PsbO in water oxidation

• Kinetics of reactions in the PSII induced by the third flash in D2O

based solutions with and without Cah3/BC together with substrate

exchange experiments to elucidate the role of BC in water oxidation

• Docking site of Cah3 protein and possible regulation of its binding

• The source of carbonic anhydrase activity in higher plant PSII

41

• The light induced structural changes of the PsbO protein both in the

PSII preparations and in vivo

• Role of CCG in the water splitting by means of mutagenesis

• The regulation of water supply by dynamics of the PsbO

IV. Acknowledgements

First and foremost, I would like to thank my supervisor Göran Samuelsson

for his understanding, as well as support and the opportunities through all

these years and my co-supervisor Bertil Andersson for exciting discussions.

I would like to express my gratitude to all collaborators and co-authors that

have added input to this work: Wolfgang Junge, Holger Dau, Vyacheslav

Vasilevich Klimov and members of his lab in Pushchino and to Bjorn Martin,

Leszek Kleczkowski and Alex Ivanov for fruitful discussion on this thesis, and

for teaching experience. I would like to thank Jim Wallgren for his help with

the printing.

My personal thanks to Anita Erkki, Eva Selstam for their friendship.

I am thankful to all present and former members of FysBot and to all

members of Russian community in Umeå for creating the warm atmosphere.

It has been a joy to be here, working together with you.

Last but not least, I would like to thank my lovely family Peter and Pavel.

42

43

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