phd thesis afj van aken

Effects of the expression of alternative oxidase on

oxidising pathway kinetics in Schizosaccharomyces pombe

mitochondria

Alexander Frans Johan van Aken

Submitted for the degree of Doctor of Philosophy

University of Sussex

July 2006

ii

I hereby declare that this thesis has not been and will not be submitted in whole or in part to

this or any other University for a degree.

Signed……………………………………………

iii

Acknowledgements

I would like to express my gratitude to professor Moore for giving me the opportunity to do

a D.Phil. project in his laboratory which has been a bumpy ride at times but was overall a

fruitful experience. Although I started working in a different field, having returned to

neuroscience I still manage to find the time to further explore my bioenergetic research and

being a tutor. I would like to thank the (past) members of the Moore laboratory (Charles,

Jane, Paul, Alice, Sarah, Rob, Nick) for their help and support over the years. I would like

to particularly thank Dr Mary Albury for her supervision with regards to the yeast

expression system. I would also like to thank Dr David Whitehouse for helping me

improve my English scientific writing and for having many useful scientific discussions. I

would also like to thank professor Derek Lamport for many not so useful scientific

discussions. Many thanks also to professor Kros for his scientific support.

I would especially like to thank Judita a fellow PhD sufferer over the years for all the

support and having spent several years together in suspended animation (hvala lijepa moja

učiteljica). Many thanks to my parents and sister for the financial support and for putting up

with not seeing me very much over the past four years. Also many thanks to Remon and

Angelique for helping out financially at times. Many thanks to all the lovely people I met

and am still in contact with after having lived in Kings Road for all the good times and for

many visits to Poland, Croatia and Slovakia (Maja, Zuzana, Wojciech, Przemek, Ewa,

Basha, Vasek, Marek, Zuzana, Sandra, Tamara, Hidemi, and Gang) hvala, d’akujem and

dziękuję.

iv

Summary

The alternative oxidase (AOX) is a non-protonmotive terminal oxidase found in the

respiratory chains of higher plants, various fungi and some protists. Its activity results in

dissipation of free energy and affects efficiency of energy transduction in mitochondria. A

plant AOX has been heterologously expressed previously in Schizosaccharomyces pombe

mitochondria in this laboratory. The work presented in this thesis describes the effects of

the expression of AOX on the respiratory kinetics of isolated yeast mitochondria.

Succinate dehydrogenase (SDH) is the only respiratory complex which is both a component

of the electron transfer chain and the citric acid cycle and possible has a strong regulatory

role. It is still relatively unknown how this enzyme is regulated exactly.

SDH in potato mitochondria can be activated by ADP, ATP and oligomycin. It has

been hypothesized that these substances activate SDH indirectly via an effect on the

membrane potential. This hypothesis was tested in a series of experiments using a multi-

electrode setup.

A characterisation of S. pombe mitochondrial respiratory kinetics is given and

determinations of the membrane potential are presented for the first time.

Oxidising pathway kinetics of S.pombe mitochondria are notably different from

what is seen in mitochondria from other tissues. Results indicate that cytochrome bc1

complex activity is probably the underlying mechanism responsible for these kinetics.

The expression of AOX in S. pombe mitochondria showed a substrate dependent

difference in oxidising pathway kinetics. It was determined that neither cytochrome

pathway or alternative pathway activity could account for these differences.

v

Contents

Acknowledgements iii

Summary iv

Contents v

Abbreviations xiii

Chapter 1 General Introduction

1.1 General background 1

1.1.1 Mitochondria 1

1.1.2 Energy transducing systems 2

1.2 The electron transfer chain 7

1.2.1 General background 7

1.2.2 Complex I 9

1.2.3 Alternative NAD(P)H dehydrogenases 10

1.2.4 Complex II 11

1.2.5 Ubiquinone / Ubiquinol 15

1.2.6 Complex III 17

1.2.7 Cytochrome c 21

1.2.8 Complex IV 22

1.2.9 Complex V 22

1.2.10 Uncoupling protein 23

1.2.11 Alternative oxidase 24

1.3 Schizosaccharomyces pombe 30

1.3.1 General background 30

1.3.2 The respiratory chain of S. pombe mitochondria 32

vi

1.3.3 SDH activation in S. pombe mitochondria 34

1.4 Summary energy transducing systems 35

1.5 A modular representation 36

1.6 Summary 38

1.7 AIMS 38

Chapter 2 Materials and Methods

2.1 Isolation and purification of mitochondria 40

2.1.1 Schizosaccharomyces pombe 40

2.1.1.1 The Expression system 40

2.1.1.2 Yeast transformation 41

2.1.1.3 S. pombe growth 41

2.1.1.4 Isolation of mitochondria from S. pombe cultures 43

2.1.1.5 Spheroplast preparation 43

2.1.1.6 Isolation of mitochondria 43

2.1.1.7 S. pombe media 44

2.1.2 Saccharomyces cerevisiae 46

2.1.3 Potato tuber 46

2.1.3.1 Isolation of mitochondria from potato tubers 47

2.1.3.2 Potato tuber media 47

2.1.4 Arum maculatum 48

2.1.4.1 Isolation of mitochondria from Arum maculatum spadices 48

2.1.4.2 Arum maculatum media 49

2.1.5 Specifics of plant mitochondrial isolation 50

2.2 Polyacrylamide gel electrophoresis & Western analysis 50

2.2.1 SDS-PAGE 50

2.2.2 Blotting to nitrocellulose 50

2.2.3 Immuno-detection of proteins 51

2.3 Protein estimations 51

2.4 Electrochemical techniques 52

vii

2.4.1 The oxygen electrode 53

2.4.2 The Q-electrode 54

2.4.3 The TPP+-electrode 57

2.4.3.1 The TPP+-electrode setup 59

2.4.3.2 Detection of [TPP+

o] 60

2.4.3.3 Construction of the TPP+-electrode membrane 60

2.4.3.4 Conditioning of the TPP+-electrode 61

2.4.3.5 Calibrating the TPP+-electrode 62

2.4.3.6 TPP+-electrode correction 63

2.4.3.7 TPP+-electrode sensitivity 63

2.4.3.8 Durability of TPP+-electrodes 64

2.4.3.9 TPP+-electrode response time 64

2.4.4 Respiratory measurements 65

2.4.4.1 Preparation of respiratory effectors 65

2.4.4.2 Nomenclature 66

2.4.4.3 Basic bioenergetic parameters 67

2.4.4.4 Q-pool kinetics 67

2.4.5 Modelling of Q-pool data 68

2.6 Other methods 70

2.6.1 Spectroscopy 70

2.7 Bioinformatic resources 70

Chapter 3 New insights into the regulation of plant succinate

dehydrogenase - revisited

3.1 INTRODUCTION 71

3.1.1 General background and aims 71

3.1.2 The membrane potential in mitochondria 73

3.1.3 Regulation of SDH 75

3.2 RESULTS 77

viii

3.2.1 General characterization 77

3.2.2 Stimulation of SDH by adenine nucleotides 81

3.2.3 Are the effects of ATP on , Qr/Qt and vO2 simultaneous? 86

3.2.4 Stimulation of SDH by adenine nucleotides in the presence of

uncoupler

89

3.2.5 Stimulation of SDH by oligomycin 91

3.2.6 Adenine nucleotides and oligomycin inhibit succinate dependent

respiration in potato mitochondria

93

3.3 DISCUSSION 99

Chapter 4 Respiratory characteristics of

Schizosaccharomyces pombe mitochondria

4.1 INTRODUCTION 112

4.2 RESULTS 113

4.2.1.1 Respiratory rates with different substrates 113

4.2.2 Schizosaccharomyces pombe - cytochrome pathway kinetics 122

4.2.2.1 The relationships of Qr/Qt vs. vO2 and ∆ vs. vO2 under ADP

limited conditions with NADH as a substrate

122

4.2.2.2 The relationships of Qr/Qt vs. vO2 and ∆ vs. vO2 under state 3

conditions with NADH as a substrate

124

4.2.2.3 The relationship between Qr/Qt vs. vO2 under uncoupled

conditions with NADH as a substrate

127

4.2.2.4 A comparison of NADH and succinate dependent Qr/Qt vs. vO2

and ∆ vs. vO2 relationships under ADP limited conditions

130


relationships under state 3 conditions

131


relationships under uncoupled conditions

132

4.2.3 Schizosaccharomyces pombe - reducing pathway kinetics 134

4.2.3.1 SDH reducing pathway kinetics in sp.011 wt mitochondria under

state 2 and uncoupled conditions

134

ix

4.2.3.2 External NADH dehydrogenase reducing pathway kinetics in sp.011

wt mitochondria under state 2 and uncoupled conditions

136

4.2.4 Are the biphasic patterns due to cytochrome bc1 complex kinetics? 137

4.2.5 Are biphasic respiratory kinetics a characteristic of yeast mitochondria? 141

4.3 Discussion 142

4.3.1 Respiratory characteristics of S. pombe mitochondria 142

4.3.2 Are the biphasic patterns due to an experimental artefact? 146

4.3.3 Are the biphasic patterns due to cytochrome bc1 complex kinetics? 147

4.3.4 Are biphasic respiratory kinetics a characteristic of yeast mitochondria? 148

Chapter 5 Functional expression of the alternative oxidase in



5.2 RESULTS 153

5.2.1 General characterisation of sp.011 AOX, AOX + T, pREP and wt

respiratory kinetics

153

5.2.2 Oxidising pathway kinetics with NADH as substrate 157

5.2.2.1 Comparing sp.011 pREP and wt cytochrome pathway kinetics with

NADH as substrate

158

5.2.2.2 Comparing sp.011 AOX and sp.011 AOX + T oxidising pathway

kinetics with NADH as a substrate

163

5.2.3 Oxidising pathway kinetics with succinate as substrate 170

5.2.3.1 Comparing sp.011 AOX and sp.011 AOX + T oxidising pathway

kinetics with succinate as substrate

170

5.2.4 Oxidising pathway kinetics in sp.011 AOX mitochondria 176

5.2.4.1 Comparing sp.011 AOX oxidising pathway kinetics with either

NADH or succinate as a substrate

176

5.2.5 Cytochrome pathway kinetics in sp.011 AOX+T mitochondria 179

5.2.5.1 Comparing sp.011 AOX + T cytochrome pathway kinetics with

either NADH or succinate as a substrate

179

5.2.6 Alternative pathway kinetics in sp.011 AOX mitochondria 181

x

5.2.6.1 Comparing sp.011 AOX alternative pathway kinetics with either

NADH or succinate as substrate

181

5.2.7 Oxidising pathway kinetics in Arum maculatum mitochondria 183

5.2.7.1 Investigating substrate dependent differences in oxidising pathway

kinetics in Arum maculatum mitochondria

183

5.3 DISCUSSION 191

5.3.1 Differences between the various S. pombe mitochondria used 191

5.3.2 Does transformation of S. pombe mitochondria lead to changes in

respiratory kinetics?

192

5.3.3 Comparing sp.011 AOX and sp.011 AOX+T oxidising pathway

kinetics with NADH as a substrate

192

5.3.4 Does AOX activity affect in S. pombe mitochondria? 193

5.3.5 Comparing sp.011 AOX and sp.011 AOX+T oxidising pathway

kinetics with succinate as a substrate

195

5.3.6 Why are the oxidising pathway kinetics obtained in this study different

from Affourtit’s study?

196

5.3.7 Are there any substrate dependent differences in sp.011 AOX oxidising

pathway kinetics?

196

5.3.8 Are the substrate dependent differences a characteristic of the

cytochrome pathway?

197


alternative pathway?

197


expression system used?

198

5.3.11 Can the alternative pathway compete with the cytochrome pathway in

S. pombe mitochondria expressing AOX?

198

5.3.12 Does expression of the alternative oxidase lead to a change in Q-pool

behaviour?

198

5.4 CONCLUSION 199

xi

Chapter 6 Modelling of oxidising pathway kinetics in

Schizosaccharomyces pombe mitochondria expressing the alternative

oxidase


6.1 RESULTS 201

6.1.1 Modelling of sp.011 AOX oxidising pathway kinetics 201

6.1.2 Are the oxidising pathway activities additive? 204

6.1.3 sp.011 AOX mixed titration studies 207

6.1.4 Applying Q-pool kinetics to fit sp.011 AOX mixed substrate oxidising

pathway data

212

6.3 DISCUSSION 215

6.4 CONCLUSION 222

Chapter 7 General Discussion

7 General Discussion 223

7.1 Characterisation of the wild type S. pombe mitochondria 224

7.1.1 How does cytochrome bc1 complex activity lead to biphasic

cytochrome pathway kinetics in S. pombe mitochondria?

225

7.1.2 Future work suggestions pertaining to the biphasic patterns in S. Pombe

cytochrome pathway kinetics

230

7.2 Functional expression of AOX in S. pombe mitochondria

yields substrate dependent differences in oxidising pathway

kinetics

231

7.2.1 Does AOX activity affect in S. pombe mitochondria ? 232

7.2.2 Substrate dependent differences in oxidising pathway kinetics of S.

pombe mitochondria expressing AOX

232

7.2.3 What causes the substrate dependent differences in oxidising pathway

kinetics in S. pombe mitochondria expressing AOX?

233

7.2.4 Are the substrate dependent differences in oxidising pathway kinetics 234

xii

in S. pombe mitochondria expressing AOX due to dehydrogenase

characteristics?

7.2.5 Future work suggestions pertaining to the substrate dependent oxidising

pathway kinetics in S. pombe mitochondria expressing AOX

236

7.3 Conclusion 237

Appendix 1 238

Appendix 2A 241

Appendix 2B 242

References 244

xiii

Abbreviations

H

~

proton electrochemical gradient

membrane potential

G Gibbs free energy

p protonmotive force

AA antimycin A

ADH alcohol dehydrogenase

ADP adenosine 5'-diphosphate

AK adenylate kinase

AMP adenosine 5'-monophosphate

ANC adenine nucleotide carrier

AOX alternative oxidase

ATP adenosine 5'-triphosphate

BCA bicinchoninic acid

BSA bovine serum albumin

CAT carboxyatractyloside

CCCP carbonyl cyanide m-chlorophenylhydrazone

DCCD N,N’-dicyclohexylcarbodiimide

DCIP 2,6-dichlorophenolindophenol

DNP 2,4-dinitrophenol

E°

standard redox potential

ETC electron transfer chain

G6P Glucose-6-Phosphate

G6PD Glucose-6-Phosphate dehydrogenase

F Faraday constant (9.65104 C mol

-1)

FAD flavin adenine dinucleotide

FMN flavin mono-nucleotide

ISP iron-sulfur protein

IMM inner mitochondrial membrane

xiv

IMS intermembrane space

KCN potassium cyanide

NADH nicotinamide adenine dinucleotide, reduced form

OAA oxaloacetate

OG octyl gallate

OMM outer mitochondrial membrane

MK menaquinone

Pi inorganic phosphate

pmf protonmotive force

PmitoKATP plant mitochondrial K+

ATP channel

PMS phenazine methosulfate

Q ubiquinone

QH2 ubiquinol

Qr/Qt Q-redox poise

R gas constant (8.31 J mol-1

K-1

)

RCR respiratory control ratio

ROS reactive oxygen species

SDH succinate dehydrogenase

SHAM salicyl hydroxamic acid

SMP submitochondrial particle

SQOR succinate:quinone oxidoreductase

T absolute temperature (K)

TPP+

tetraphenyl phosphonium

UCP uncoupling protein

vO2 oxygen consumption rate

z valence number

1

Chapter 1

General introduction

1.1 General background

1.1.1 Mitochondria—Mitochondria are double walled organelles found in eukaryotic cells

(Figure 1.1). The innermost compartment, the matrix, is separated from the intermembrane

space (IMS) by the inner mitochondrial membrane (IMM) which is relatively impermeable

to ions and large solutes. The outer mitochondrial membrane (OMM) on the other hand is

relatively permeable to most solutes with a molecular weight less than 10 kDa [1] and

because of this the intermembrane space is assumed to be continuous with the cytosol. The

IMM shows numerous invaginations (cristae). Whether or not the cristae are continuous

with the intermembrane space is still under investigation [2]. In this study it is assumed that

under in vitro conditions (isolated mitochondria in solution) there are only two

compartments, the matrix and outside of the matrix.

Figure 1.1 A schematic representation of a mitochondrion. (Source: courtesy of Dr. Michael W. Davidson,

Florida State University). Mitochondria are typically depicted in this ‘sausage’ form but the shape can vary

dramatically depending on tissue and/or developmental state.

2

Mitochondria are known as the powerhouses of cells responsible for the generation of ATP,

the cellular energy currency. ATP is used to drive many thermodynamically unfavourable

processes in the cell and a continuous supply is needed in order for the cell to survive. For

instance, to continuously maintain a resting membrane potential most cells expend as much

as 30% of cellular ATP to keep the Na+/K

+ exchanger active, neurons in the central nervous

system have to expend as much as 70% [3]. A healthy complement of mitochondria is

therefore vital for cellular functioning.

The energy released upon hydrolysis of the terminal anhydride bond of the ATP molecule

is used to drive the uphill process to which ATP hydrolysis is coupled. Hydrolysis of one

mole of ATP under normal cellular conditions, i.e. where the mass action ratio of the ATP

synthesis reaction is kept away 10 orders of magnitude from equilibrium, can release 57 kJ

of energy. The ratio of ATP to ADP concentration in the cytosol is typically maintained at a

value of 1000 [1]. Mitochondria do not just generate ATP at a constant rate, ATP synthesis

is tightly regulated and mitochondrial activity is highly flexible depending on energetic

conditions.

Given that cells rely heavily on the efficient generation of ATP by the mitochondria it is

curious that respiratory chains of many organisms contain respiratory complexes which

actively reduce this efficiency by dissipating energy. Two of these complexes are the

uncoupling protein (UCP) (see section 1.2.10) and the alternative oxidase (AOX) (see

section 1.2.11). In order to understand how these complexes can reduce the efficiency with

which ATP is synthesized an understanding of energy transducing systems is needed.

1.1.2 Energy transducing systems—Returning to the example of neurons, the brain needs a

continuous supply of oxygen and glucose; temporary shortages of either of them (e.g.

ischaemia) can lead to disastrous results in which cells may go into apoptosis. Upon

restoring the supply of oxygen and glucose things may get even worse as happens under the

conditions of reperfusion injury [4] and excitotoxicity [3], during which mitochondrial

processes set off a series of unfortunate events which lead to cell death. During oxygen

deprivation mitochondria become ‘highly reduced’; when oxygen becomes available again

this increased reduction level leads to the formation of reactive oxygen species (ROS)

which leads to the breakdown of membranes jeopardizing cellular integrity.

3

In order to understand how the synthesis of ATP, the consumption of glucose and oxygen

and the formation of ROS are related a brief description of energy transducing systems will

be given.

All organisms need a continuous energy supply in order to prevent a state of

thermodynamic equilibrium (death). Energy is readily available in the form of

electromagnetic rays from the sun for those organisms which can trap this form of energy

to subsequently transduce it into another form. Most other organisms derive energy from

the breakdown of ‘energy rich’ compounds, such as glucose.

The breakdown of glucose under standard conditions yields 2870 kJ mol-1

. Most energy

utilising reactions in the cell require between 10 to 50 kJ mol-1

[5] so there is a need to

partition the energy released during breakdown of glucose. ATP, releasing 57 kJ mol-1

upon

hydrolysis (under cellular conditions) is used predominantly. Some ATP is generated

through substrate level phosphorylation (about 5% [6]), in the presence of molecular

oxygen however the bulk of cellular ATP is generated via energy transducing reactions.

Basically all energy transducing systems operate along the same principles: two proton

pumps, located in the same membrane (which is relatively impermeable to protons and

other ions) their activities coupled to each other via a proton current. In Figure 1.2 the

situation as it occurs in mitochondria is shown schematically. By convention the matrix is

considered the N side (N for negative) and the intermembrane space the P side (P for

positive). The so called primary pump utilises electrons1 to drive the transport of protons

against their concentration gradient from the matrix to the intermembrane space.

This creates a protonmotive force (pmf) which is subsequently utilised by the secondary

pump to drive the synthesis of ATP via the influx of protons from the intermembrane space

to the matrix.

1 It would be more correct to use the term ‘reducing equivalents’ as will be explained further in section

1.2.

4

Figure 1.2 A schematic representation of an energy transducing membrane containing two proton pumps

communicating with each other via a proton circuit. N: negative P: positive.

The pmf, or p, is a driving force with units of V, which consists of two components: a

concentration gradient of protons (pH) and an electrical potential difference ().

Displacement of ions across a membrane generates an electrochemical potential which is

expressed in kJ mol-1

(units of energy).

The change in free energy (G) upon the transport of 1 mol of protons across a membrane

(in the absence of a ) is given by the following equation:

[1.1]

R: gas constant (8.31 J mol-1

K-1

)

T: absolute temperature (K)

i: inside

o: outside

o

i

H

HRTmolkJG

][

][log3.2)1 (

5

The free energy change associated with the separation of 1 mol of univalent ions across a

membrane (in the absence of a concentration gradient) is given by:

zFmolkJG )1 ( [1.2]

z: valence number (1 in this case)

F: Faraday constant (9.65104 C mol

-1)

Protons in the matrix and the intermembrane space normally will be affected by both a

concentration gradient and an electrical gradient which gives:

o

i

H

HRTzFmolkJG

][

][log3.2)1 ( [1.3]

This Gibbs energy difference is generally referred to as the proton electrochemical gradient:

H

~

And with the definition for pH (pH = - log [H+]) the equation can be further simplified:

pHRTFmolkJH 3.2) (

~1 [1.4]

To facilitate comparison with redox potential differences in the electron transfer chain

(ETC) Mitchell [1] defined the term protonmotive force (p) which is:

FmVp H /)( ~

[1.5]

6

p is expressed in units of V and substituting values for R and T at 25 C the equation

simplifies to:

pHmVp 59)( [1.6]

A good understanding of these basic equations is necessary to appreciate the method with

which membrane potentials were determined in this study. This topic will be discussed in

detail in chapter 2.

7

1.2 The electron transfer chain

1.2.1 General background—Figure 1.3 shows the ETC as it is organised in the

mitochondria of mammals. The various components within the chain are organised

according to their redox potentials in order of increasing value. Substrates (e.g. NADH or

succinate) can be oxidised at specific locations where they donate reducing equivalents (a

reducing equivalent can be defined as 1 mole of hydrogen atoms, one proton and one

electron per H atom [6]). The red arrows indicate the transfer of electrons through the

chain, which eventually reduce oxygen to water at complex IV. At three sites (complexes I,

III and IV) the transfer of electrons is coupled to the translocation of protons from the

matrix to the intermembrane space (blue arrows), this generates the aforementioned pmf,

the matrix being negative with respect to the IMS. Protons can re-enter the matrix via

complex V, a process which is coupled to the synthesis of ATP from ADP and Pi. Another

inward pointing arrow indicates the passive leak of protons back into the matrix, it is

postulated that protons can traverse the IMM via the junctions between lipid and protein

[1]. Apart from leak and ATP synthesis there are many transporters (symporters and

antiporters) which utilise the proton electrochemical gradient to drive the translocation of

metabolites across the IMM (not shown in the figure). Overall, oxygen is consumed and

substrates are oxidised, as a result of this, energy is stored in a pmf, which can be utilised

by the ATP synthase to drive the reaction of ATP synthesis, this process is known as

oxidative phosphorylation.

The components of the mammalian ETC are: complexes I to V, the Q pool and

cytochrome c. With respect to the relative abundance of complexes within the ETC the

following stoichiometry is currently accepted: complexes I : II : III : IV : cytochrome c :

ubiquinone = 1 : 2 : 3 : 7 : 14 : 63 [7]. The plant ETC contains the same components but is

more complicated than its mammalian counterpart due to the presence of some extra

respiratory proteins, see Figure 1.4. The plant ETC contains several alternative NAD(P)H

dehydrogenases, which are non-protonmotive, two of them located on the inner leaflet of

the IMM and two on the outer leaflet. Another component is the alternative oxidase, which

like complex IV catalyses the reduction of molecular oxygen to water [8].

8

Figure 1.3 Schematic representation of the mammalian ETC. I : complex I (NADH dehydrogenase),

II : complex II (succinate dehydrogenase), III: complex III (ubiquinol:cytochrome c oxidoreductase),

IV: complex IV (cytochrome c oxidase), V: complex V (ATP synthase), c: cytochrome c, Q:

the Q-pool (ubiquinone + ubiquinol). Blue arrows: proton flow. Red arrows: electron flow. Also

indicated is the non specific leak of protons across the IMM.

The route taken by electrons transferred from QH2 to complex III (and subsequently to

cytochrome c to complex IV) is referred to as the cytochrome pathway. Electrons

transferred to AOX are said to use the alternative pathway. The main difference between

these two pathways is that the alternative pathway is non-protonmotive [8].

Figure 1.4 Schematic representation of the plant ETC which contains several additional components

compared to the mammalian system (see Figure 1.3). NDH (non-protonmotive NADH dehydrogenase), AOX

(alternative oxidase).

9

A physical description of the components of the ETC will be given in the remainder of this

section. The alternative oxidase will be discussed in detail given its importance in this

study. Also complexes II and III will be discussed in somewhat more detail because a

thorough understanding of the functioning of these respiratory proteins is necessary in order

to interpret the acquired experimental results.

1.2.2 Complex I (NADH:quinone oxidoreductase, NADH dehydrogenase):

Complex I catalyses the transfer of two electrons to ubiquinone in a

reaction coupled to proton translocation across the IMM. Currently

the proton translocation stoichiometry is believed to be 4H+/2e

- [1].

Of all the complexes involved in oxidative phosphorylation, complex I is

by far the largest. In mammalian mitochondria it consists of 43 subunits with a total

molecular weight in the range of 750-1000 kDa. Not all of these subunits are required for

electron transfer as it was found that bacteria contain a minimal functional unit of just 14

subunits [1]. Complex I is normally taken to be L-shaped with a hydrophilic and a

hydrophobic part. The hydrophilic part contains a flavin mono-nucleotide (FMN) moiety

which is reduced by NADH, electrons subsequently are transferred through 8 or 9 iron

sulfur clusters (FeS) where a molecule of ubiquinone (Q) accepts the electrons. Complex I

is both nuclear (nDNA) and mitochondrial (mtDNA) encoded and is potently inhibited by

rotenone, piericidin A [9] and rhein [10]. Recently, complex I defects caused by pathogenic

mutations in mtDNA and nDNA have been linked to various neurodegenerative diseases

such as Parkinson’s disease [9]. Defective complex I functioning leads to a decreased H+

and a concomitant decrease in ATP production whereas ROS formation is stimulated.

10

1.2.3 Alternative NAD(P)H dehydrogenases [11, 12] : In mammalian mitochondria the only

ETC complex able to accept reducing equivalents from NADH is complex I. In the

mitochondria of plants and fungi (including S. pombe) one or more alternative NAD(P)H

dehydrogenases can be found. Like complex I these dehydrogenases catalyse the transfer of

two electrons to ubiquinone, however this reaction is not coupled to proton translocation

across the IMM, therefore no energy is conserved. Another difference is the use of a flavin

adenine dinucleotide molecule (FAD) as a redox prosthetic group instead of FMN. The

external NADH dehydrogenase (Ext. NDH) and the external NAD(P)H dehydrogenase

(Ext. N(P)DH) are situated at the outer leaflet of the IMM facing the IMS. The internal

NADH dehydrogenase (Int. NDH) and the internal NAD(P)H dehydrogenase (Int. N(P)DH)

are situated at the inner leaflet of the IMM facing the matrix. Unlike complex I all the

alternative NADH dehydrogenases are believed to be relatively small with only one to four

subunits. Complex I inhibitors have no effect on the alternative NAD(P)H dehydrogenases

and any inhibitors which do affect these complexes are rare and mostly unspecific. It is

hypothesized that alternative NADH dehydrogenases can be employed as a dynamic

response to changing metabolic needs. Given their small size they can be made readily

available as opposed to complex I which requires 43 subunits to be expressed. Varied

expression and activity of the alternative NAD(P)H dehydrogenases and the alternative

oxidase provides flexibility in regulating the redox state of cytoplasmic and mitochondrial

matrix NAD(P)H pools. Mitochondria of some organisms lack complex I completely (e.g.

S. cerevisiae and S. pombe [13]) and they are dependent on alternative NADH

dehydrogenases to oxidise matrix generated NADH.

External NADH dehydrogenase: The Ext. NDH dependent oxygen uptake can be

stimulated by the presence of divalent cations which electrostatically screen negative

membrane charges. Also Ext. NDH has a high affinity for calcium binding which is

believed to affect the interaction with ubiquinone. Early work done on external NADH

oxidation gave ADP/O* values between 1.2 and 1.4 whilst NADH oxidation could be

inhibited with antimycin A (AA, complex III inhibitor) and cyanide (complex IV inhibitor).

These observations indicate that electrons enter the ETC just before complex III. Its

molecular weight is estimated to be 32 kDa.

* the amount of ADP molecules converted to ATP molecules per atom of oxygen , see section 2.4.4.3.

11

External NAD(P)H dehydrogenase: The Ext. N(P)DH has similar ADP/O values as

the Ext. NDH and it is also inhibited by AA and cyanide indicating a point of entry in the

ETC just before complex III. The Ext. NDH and Ext. N(P)DH have different pH profiles.

Also Ext. N(P)DH is more calcium dependent than Ext. NDH. A protein doublet with

molecular weight 58 kDa, localized to the outer surface of the IMM, was found to oxidize

both NADH and NADPH.

Internal NADH dehydrogenase: Internal NADH oxidation, in the presence of

rotenone, showed ADP/O values of 1.5 in plant mitochondria. It was also found that the Int.

NDH had a ten times lower affinity for NADH than complex I. No calcium activation has

been found so far. In S. cerevisiae mitochondria a single polypeptide with a weight of 53

kDa was identified as an internal NADH dehydrogenase.

Internal NAD(P)H dehydrogenase: Unlike the Int. NDH the Int. N(P)DH is

activated by calcium. Apart from NAD(P)H it possibly also oxidizes NADH.

Its molecular weight is estimated to be 43 kDa.

A complex identified as an NADPH dehydrogenase in one species may be found in another

species where it can only oxidise NADH, this illustrates that the alternative NADH

dehydrogenases still require a lot of research.

1.2.4 Complex II (succinate dehydrogenase) [14, 15]:

Succinate dehydrogenase (SDH) is the only ETC complex which is also a component of the

citric acid cycle and fulfils therefore a dual role, being active in both the process of energy

transduction and the generation of carbon intermediates for biosynthetic metabolism. SDH

is a member of the succinate:quinone oxidoreductases (SQOR, EC 1.3.5.1). SQORs couple

the oxidation of succinate to fumarate to the reduction of quinone to quinol [16]:

succinate fumarate + 2H+ + 2e

-

quinone + 2H+ + 2e

- quinol

12

This oxidoreduction reaction is not coupled to proton translocation therefore complex II

does not contribute to the conservation of energy. In vitro, SQORs can catalyse both

succinate oxidation and fumarate reduction, be it at different rates. By providing substrate

in excess, directionality is achieved under experimental conditions. SQORs consist of four

subunits referred to as A, B, C and D, see Figure 1.5.

Figure 1.5 Succinate dehydrogenase. The hydrophilic subunits A and B are exposed to the matrix (negative

side). The hydrophobic subunits C and D are situated within the IMM. The SDH shown here is a type C

SQOR (class 3) which is normally found in eukaryotic mitochondria. Adapted from Figure 2C in [14].

The presence of a single heme group (indicated by the rectangle within subunits C and D)

and the presence of two hydrophobic subunits are indicative of an eukaryotic SDH.

Other classes show variations in the amount of heme groups and hydrophobic subunits.

Another way of classifying SQORs is on the basis of quinone substrate. In this study the

respiratory activity of yeast and plant mitochondria was investigated therefore no

description of archeal and bacterial SQORs will be given here, for more information on

these complexes see Refs 14 and 15. Although most bacteria express SDH of a form

different from what is found in eukaryotic species, recently acquired X-ray structures show

that SDH in E. coli would be classified equivalent to the mammalian complex [16, 17], see

Figure 1.6.

13

Figure 1.6 Three dimensional structure of the E. coli SDH taken from Figure 1C in [17]. Subunits A and B

are coloured teal and purple respectively. Subunits C and D are shown in orange and yellow respectively.

Prosthetic groups shown are covalently bound FAD (subunit A), [2Fe-2S], [4Fe-4S] and [3Fe-4S] iron-sulfur

centers (subunit B). Subunits C and D display bound quinone (black) and heme b556 (magenta). The E. coli

SDH is equivalent to the SQOR type normally found in eukaryotic mitochondria.

Subunit A (also known as the flavoprotein Fp or CII-1) contains a covalently bound FAD

prosthetic group and the dicarboxylate binding site; its molecular weight is 70 kDa. Subunit

B (also known as the iron-sulfur protein or CII-2) contains three iron-sulfur clusters, [2Fe-

2S], [4Fe-4S] and [3Fe-4S] (also known as Centers 1-3) and weighs 27 kDa. Subunits C

and D (also referred to as anchor proteins or CII-3 and CII-4 respectively) contain the

quinone reduction and oxidation sites and one heme group (in eukaryotes), their molecular

14

weights are 15 and 13.5 kDa respectively. Subunits A and B have a high sequence

homology amongst species, whereas this is much lower for subunits C and D. All subunits

are nuclear encoded making complex II unique in the sense that the other main ETC

complexes (I, III, IV and V) are all partially encoded by mitochondrial DNA. Subunits A

and B are hydrophilic and extend into the matrix. Subunits C and D are hydrophobic and

span the IMM, i.e. parts of subunits C and D are accessible from the cytosolic side.

Electron transfer though complex II is linear and experimental data have shown that

electron transfer through SDH is not sensitive to uncouplers [17]. Succinate binds to the Fp

unit where it subsequently donates two electrons and two protons to the FAD group

reducing it to FADH2. From there electrons are transferred to the IP unit where they pass

through the three iron-sulfur centres. The electrons end up reducing ubiquinone to

ubiquinol where two protons are taken up from the matrix. At present the role of heme in

the electron transfer from succinate to ubiquinone is unclear.

Inhibitors:

Malonate and oxaloacetate (OAA) are potent competitive inhibitors of SDH. Several

inhibitors interfere with quinone binding such as 2-thenoyltrifluoroacetone (TTFA) and 3-

methyl-carboxin and 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO). These inhibitors do

not interfere with the activity of the solubilised enzyme. The site of action is between

Center 3 and the Q-pool. Apparently cyanide disrupts Center 3 in SDH [18]. Intracellular

oxidation of succinate is inhibited by fluoride [19].

Regulation of SDH:

SDH in isolated mitochondria is in a partially deactivated state due to bound OAA [15].

The slowness of the reaction and the high energy of activation (35.6 kcal/mol*) of SDH was

interpreted as a conformational change in the enzyme [20]. SDH can be activated by ATP

and ADP [21]. Chapter 3 deals with SDH activation and regulation of SDH is discussed

more in-depth in its introduction, see section 3.1.3.

* 149 kJ/mol

15

1.2.5 Ubiquinone / Ubiquinol (Q-pool):

The electron transfer chain has two mobile pools of electron carriers: the cytochrome c pool

(see section 1.2.7) and the Q-pool. The Q-pool consists of both oxidised and reduced forms

of ubiquinone. Ubiquinone (Q) undergoes an overall 2H+ + 2e

- reduction to form ubiquinol

(QH2). Because of its long hydrocarbon side chain both Q and QH2 are highly hydrophobic

[1]. Figure 1.7 shows the chemical structure of Q. The side chain can vary in length

depending on species, n = 10 in mammalian mitochondria [1], whereas plant mitochondria

contain a mixture of ubiquinone molecules with n = 9 and 10 [22]. In yeast mitochondria n

= 6 [1]. Figure 1.8 shows the two step reduction of Q. After the first step a highly reactive

intermediate (semiquinone) is formed, if allowed to react with molecular oxygen it would

lead to ROS formation. Hence the necessity to firmly bind semiquinone during reduction of

Q to QH2 as will be discussed in 1.2.6. It is generally assumed that the Q-pool functions as

a homogenous pool of electron carriers which forms the linkage between dehydrogenases

(complexes I and II and the alternative NADH dehydrogenases) and ubiquinol oxidases

(complex III and AOX). In mammalian submitochondrial particles (SMP) the activity of

both complex I and II were found to be linearly dependent on the Q redox poise (QH2 /

(Q+QH2)). A linear relationship was also found between the dependency of complex III

activity on Q redox poise. These linear dependencies from both dehydrogenases and

ubiquinol oxidases on the level of Q reduction are commonly referred to as quinone pool

behaviour [23, 24].

Figure 1.7 Structure of ubiquinone as adapted from Figure 5.6 in [1]. The length of the side chain (R) can

vary, n = 10 in mammalian mitochondria, in plant mitochondria a mixture of n=9 and n=10 is found. n = 6 in

yeast mitochondria2 and n = 9 in amoebal mitochondria.

2 This figure does not hold for all yeasts, e.g. Candida utilis mitochondria contain both UQ7 and UQ9 [25].

16

Quinone Semiquinone Quinol

Figure 1.8 Two step reaction of quinone reduction to ubiquinol.

The homogeneity of the Q-pool was deduced from the observation that antimycin A

titrations (which inhibit complex III) do not result in a linear response. Figure 1.9 shows a

plot with fictitious data representing two situations: one in which every Q molecule is in a

fixed relationship with a bc1 complex () and one in which all Q molecules are free to

engage with different bc1 complexes (). In the first situation inhibition titrations with AA

would lead to a linear relationship. In the second situation Q molecules can still donate

electrons to uninhibited bc1 complexes and overall electron flux, expressed as respiratory

activity is seen to be ‘antimycin resistant’. The antimycin resistant respiratory kinetics are

normally seen in mitochondria [24, 26]. In an article on pool behaviour of Q (and

cytochrome c) in S. cerevisiae mitochondria Boumans et al. [27] reported non-homogenous

pool behaviour and a linear relationship between respiratory activity and complex III

inhibition was found; from this it was concluded that in yeast mitochondria the respiratory

components of the ETC are arranged as an ordered macromolecular assembly which does

not allow for diffusion based collisions between components. Another study done in the

same year by Rigoulet et al. [28] showed that S. cerevisiae mitochondria do show

homogenous pool behaviour (cf. Figure 3B in [28]). Several studies showed that AOX

(from either Candida albicans or Lycopersicon esculentum (tomato)) expressed in S.

cerevisiae mitochondria [29] and [30] respectively, could utilise QH2 as a substrate, which

implies Q-pool behaviour in S. cerevisiae.

e- e

- + 2H+

17

Figure 1.9 Theoretical antimycin A titration data points illustrating homogenous () and non-homogenous

Q pool () behaviour in mitochondria, see text for details.

1.2.6 Complex III (ubiquinol:cytochrome c oxidoreductase, bc1 complex):

The bc1 complex is a protonmotive homodimer catalysing the oxidation of ubiquinol to the

reduction of cytochrome c. In bovine heart and S. pombe mitochondria each monomer

consists of 11 subunits of which 8 do not have a catalytic role in the oxidation of ubiquinol

[31]. Complex III in S. cerevisiae mitochondria contains 10 subunits. A presequence

targeting the Rieske protein is cleaved from the protein; in bovine heart and S. pombe

mitochondria this cleaved presequence is retained as a subunit whereas in S. cerevisiae [31]

and in potato [32] it is degraded. The redox groups consist of a 2Fe/2S centre which is

located on the iron-sulfur protein (ISP), two B-type heme groups (bL and bH) located on a

single polypeptide and the heme of cytochrome c1 [1] (see Figure 1.10). In many bacteria a

functionally similar but structurally simpler version of the bc1 complex is found in the

plasma membrane. These complexes have the same electron transfer and proton

translocation functionality as their mitochondrial counterparts. Paracoccus for instance

18

only has a basic three subunit enzyme similar to the protein complex in Figure 1.10. This

indicates that the supernumerary subunits are not required for electron transfer or proton

translocation [31]. Crystal structures of the bc1 complex have become available in recent

years, see Figure 1.11 for the S. cerevisiae bc1 complex structure.

Figure 1.10 Schematic representation of the bc1 complex. Only the subunits containing redox groups are

shown. The iron-sulfur protein (ISP) also referred to as the Rieske protein. The b-type hemes containing

polypeptide and the cytochrome c1 subunit.

The midpoint potentials at pH 7 for the redox centres in the yeast bc1 complex are:

ISP +280 mV, cytochrome c1 +240 mV, bL –30 mV and bH +120 mV [33].

In order to understand the pathway of electron flow through the bc1 complex an

understanding of the Q-cycle [32] is needed, see Figure 1.12.

In a complete turnover of the Q-cycle two molecules of ubiquinol are oxidised, one

molecule of ubiquinone is reduced, 2 protons are taken up from the matrix, 4 protons are

released in the IMS and two cytochrome c1 groups are reduced [1]. The Q-pool in the IMM

exists in large molar excess over the bc1 complexes with a ratio of 21:1 [7].

In stage 1 a molecule of ubiquinol diffuses to the binding site Qp (p for positive as it is

situated near the positive site of the IMM) where it is oxidised in several stages:

One electron is transferred to the ISP, two protons are released to the cytosol and a

semiquinone molecule (see Figure 1.7) remains temporarily bound at Qp. The second

electron is transferred to bL. The electron transferred to the ISP passes down the ETC to

ISP cyt c1

bL

bH

IMS

matrix

19

cytochrome c1, cytochrome c and cytochrome c oxidase. The electron on bL passes onto bH.

This electron is used to reduce a molecule of ubiquinone, at another binding site Qn, to

semiquinone which remains bound there until a next molecule of ubiquinol comes along in

the second part of the cycle.

Figure 1.11 Structure of the S. cerevisiae bc1 complex taken from Figure 1A in [34].

The bc1 complex shown in its homodimeric form. Cytochrome c1 is shown in red, the Rieske protein in green,

cytochrome b in blue, the hinge domain in cyan. Antibodies binding to the bc1 complex are shown in orange.

Cytochrome c bound to one monomer is shown in yellow. All redox prosthetic groups are shown in black.

IMS: intermembrane space. IM: inner membrane. MA: matrix.

20

Figure 1.12 The Q-cycle in mitochondria, adapted from Figure 5.14 in [1]. P: positive N: negative

See text for explanation.

21

When a second molecule of ubiquinol binds to Qp some of the steps in stage 1 are repeated.

One electron is transferred to ISP, again 2 protons are released into the cytosol. One

electron is transferred to bL. The electron on ISP is passed down the ETC to cytochrome c1,

cytochrome c and cytochrome c oxidase. The electron on bL is transferred to bH. The

semiquinone molecule still bound at Qn is reduced by bH and to complete the full reduction

of semiquinone to ubiquinol 2 protons are taken up from the matrix. This completes the Q-

cycle [1].

Inhibitors:

Figure 1.12 shows two inhibition sites in the bc1 complex indicated as red bars in stage 1.

Myxothiazol blocks events at Qp and stigmatellin inhibits electron transfer to the Rieske

protein. Antimycin A acts at Qn, preventing reduction of ubiquinone by bH [1].

1.2.7 Cytochrome c:

Cytochrome c, a mobile redox carrier, is a peripheral protein located on the P-face of the

IMM which transfers electrons between complex III and IV and can be readily solubilised

from intact mitochondria. Electrons can be donated artificially from molecules such as

tetramethyl-p-phenylene diamine (TMPD) and electrons can leave the ETC via cytochrome

c through reduction of ferricyanide (Fe(CN)63-

) [1]. Cytochrome c is nuclear encoded and

has a molecular weight of about 13 kDa [35]. Apart from being an electron carrier,

cytochrome c plays a role in the process of apoptosis. Upon induction of cell death

cytochrome c leaves the confinement of the IMM and diffuses to the cytosol where it

initiates the activation of caspases, a family of cysteine proteases [35].

22

1.2.8 Complex IV (cytochrome c oxidase):

Cytochrome c oxidase in mitochondria consists of up to

13 subunits in mammalian mitochondria (11 in yeast) [36]

of which only two (subunits I and II) are involved in electron

transfer and proton translocation. Complex IV is present in the IMM as a homodimer.

The complex catalyses the complete reduction of oxygen to water and pumps protons

across the IMM with a stoichiometry of 2H+/2e

-. Four electrons are transferred sequentially

from the cytochrome c pool to complex IV. Subunit II contains a copper centre (CuA)

which has two copper ions in a cluster with sulfur atoms. This complex accepts electrons

from cytochrome c one at a time. Subunit I contains two heme groups (heme a and heme

a3) and another copper centre (CuB). Electrons from CuA are transferred to heme a onto

heme a3 and finally onto CuB where oxygen is reduced to water. Heme a3 is also the

binding site for several complex IV inhibitors: cyanide, azide, nitric oxide and carbon

monoxide [1]. A regulatory effect of adenine nucleotides on complex IV is well known,

addition of ATP to yeast mitochondria leads to an increase in enzymatic capacity of

cytochrome c oxidase but does not stimulate respiration rate [36].

1.2.9 Complex V (ATP synthase, F1.Fo-ATPase):

Complex V is a proton pump which couples the

hydrolysis of one molecule of ATP to ADP and Pi to the

translocation of three protons across the IMM. The name

F1.Fo-ATPase indicates the two mayor components of this

complex, the hydrophobic Fo complex (160kDa) which

translocates protons and the F1 complex (370 kDa) which

contains the catalytic and regulatory sites. The Fo complex is located in the IMM whereas

the F1 part extends into the matrix. The electrochemical gradient of protons generated

through ETC activity can be used to drive the synthesis of ATP and the ATP synthase is

seen to operate in reverse. The influx of protons drives the thermodynamically

23

unfavourable reaction of ATP synthesis [1] and complex V can be considered a p

consumer [37]. The Fo and F1 complexes can be targeted directly by specific inhibitors.

Oligomycin (hence the o in Fo) and venturicidin bind at the Fo complex. Aurovertin and

efrapeptin bind at the F1 complex. Dicyclohexylcarbodiimide (DCCD) has inhibitory

effects on both complexes [1].

The ATP synthase is regulated by the natural inhibitor protein IF1 which binds to a -

subunit from the F1 complex [38]. The binding interferes with the cooperative ATP

synthesis process of complex V. Binding of the protein inhibits hydrolytic activity and it is

suggested that it can also inhibit ATP synthesis [39, 40]. High p induces release of IF1

[40] and the off-rate (rate of release) is high. High concentration of ATP induces binding of

IF1 and the on-rate (rate of binding) is high under these conditions [39]. The equilibrium

between the on-rate and off-rate determines the steady state inhibition of the ATP synthase

by IF1. The IF1 protein was found in mammalian [39, 40], plant [41] and yeast

mitochondria [42]. Inhibitory proteins can cross-react with mitochondria from other sources

[38, 42].

1.2.10 Uncoupling protein:

The uncoupling proteins (UCP) are a subfamily of the mitochondrial carriers (MC) [43] of

which 5 types have been identified so far: UCP1-5. The first UCP to be found was UCP1 in

brown adipose tissue where it functions to drive non-shivering thermogenesis in

hibernators, cold-adapted rodents and newborn mammals [1].

The activity of the protein is stimulated by fatty acids and inhibited by nucleotides. By

catalysing proton transport into the matrix it dissipates the p by increasing the proton

conductivity across the IMM [43]. The p dissipation would lead to an increase in body

temperature, another beneficial purpose of stimulating UCPs would be to control ROS

production [43, 44].

24

1.2.11 Alternative oxidase (AOX):

General background:

Plant mitochondria exhibit cyanide resistant respiration to various degrees, potato tuber

mitochondria show only a little resistance to inhibition with cyanide whereas mitochondria

isolated from aroid spadix tissues seem to be fully resistant [8]. This cyanide resistant

respiration is associated with the presence of an extra terminal oxidase (apart from complex

IV) which functions as an ubiquinol:oxygen oxidoreductase and is referred to as the

alternative oxidase (AOX). It catalyses the complete reduction of oxygen to water [45].

Although generally referred to as cyanide resistant, AOX is insensitive to cytochrome

pathway inhibitors in general (e.g. antimycin A, azide and carbon monoxide). AOX is

sensitive to hydroxamic acid derivatives (such as salicylhydroxamic acid (SHAM) which

interferes with Q-binding [46]) and alkyl gallates (such as octyl gallate and dodecyl gallate

[47]). AOX accepts electrons from the Q-pool, bypassing the cytochrome pathway. Given

the non-protonmotive nature of AOX no energy is conserved during this step which is

reflected in a decreased ADP/O ratio. In a situation where reducing equivalents are donated

to either complex II (or any of the alternative NAD(P)H dehydrogenases) and if the

alternative pathway is the only available oxidising pathway, all energy freed by the

oxidation reactions is dissipated as heat. Apart from higher plants, AOX is found in several

other species which have branched respiratory pathways, such as various fungi (e.g.

Neurospora crassa and Pichia anomala [48]) and protists (e.g. Trypanosoma brucei and

Chlamydomonas reinhardtii [48]).

Quite recently (2004) the occurrence of AOX encoding genes was found to extend into the

animal kingdom as well. Sequences coding for AOX were found in the genomes of a

mollusc (Crassostrea gigas), a nematode (Meloidogyne hapla) and in chordates (Ciona

intestinalis and Ciona savignyi) [49]. The belief that AOX only occurs in eukaryotes has

been challenged recently by reports on the occurrence of AOX in prokaryotes such as

Novosphingobium aromaticivorans [50]. A recent search in a metagenomic dataset from

25

marine microbes in the Sargasso Sea uncovered 69 different AOX genes [51] which

indicates that AOX may be widespread in aquatic environments.

Cyanide resistant respiration in higher plants has been reported since the early 1900’s [52]

but only with the advent of antibodies raised against the partially purified alternative

oxidase from Sauromatum guttatum [53] was it demonstrated positively that AOX was a

genuine component of the ETC of cyanide-resistant mitochondria.

The plant alternative oxidase is nuclear encoded and the first identified gene was named

aox1 [54], importantly this gene encodes for a protein which includes a pre-sequence

targeting it to mitochondria with an approximate weight of 39 kDa. Cleaving the target

sequence yields a protein of approximately 32 kDa [55]. Several other genes (aox2a,

aox2b) have been identified since [56]. In plants, AOX is found as a homodimeric enzyme

whereas in fungi it is a monomer [57]. Apart from a structural difference both types of

AOX are also very different with respect to activation mechanisms [58]. In this study a

plant alternative oxidase from S. guttatum was heterologously expressed in S. pombe

mitochondria [59], therefore the remainder of this section will focus on plant AOX and

only significant differences between plant AOX and non-plant AOX will be discussed.

Contrasting values for the plant AOX apparent KM for oxygen have been reported from

~1.7 M to 10-20 M [26]. In any case plant AOX affinity for oxygen is lower than that of

complex IV (0.14 M [60]). The partitioning of reducing equivalents between the

alternative and the cytochrome pathway will be discussed in-depth in chapter 5.

Structure of AOX:

Several models of the AOX structure have been proposed over the years [61]. In this

section only the latest consensus model will be discussed. The current consensus model is

the Andersson Nordlund (AN) model [62]. The alternative oxidase is believed to be an

interfacial di-iron carboxylate protein [63] attached to the inner leaflet of the IMM facing

the matrix space, see Figure 1.13.

Getting structural information has been notoriously difficult and continuous efforts at

spectroscopic detection were not successful until Berthold et al. in 2002 managed to get an

EPR signal from a membrane fraction of E. coli expressing the Arabidopsis thaliana AOX

26

[52]. Their results were the first experimental evidence supporting the hypothesis that AOX

is indeed a member of the di-iron carboxylate proteins [64], a group of nonheme iron

proteins that contain a coupled binuclear iron center.

Figure 1.13 Structure of the plant AOX according to the AN model, adapted from Figure 1 in [63].

Regulation of AOX:

Regulation of the alternative oxidase can be divided into two categories, regulation through

expression and post-translational regulation. Regulation of the plant AOX is quite different

from its counterpart in fungi. For this study a plant alternative oxidase (S. guttatum) was

expressed in S. pombe, therefore in this section emphasis will be on plant AOX regulation.

Regulation through expression:

AOX expression can be increased in many ways. Stress conditions such as chilling,

wounding, injury and osmotic stress are all known to increase expression [65]. In many

fruits alternative pathway activity is known to increase during the ripening process. In

27

mango fruit alternative pathway activity, amounts of protein and mRNA levels all increase

in parallel [66]. In Hansenula anomala (now Pichia anomala) incubation with cytochrome

pathway inhibitors, such as antimycin A or KCN, led to increased transcription of AOX,

similar behaviour was seen in tobacco cells [66]. How inhibition of the cytochrome

pathway is perceived by and transmitted to the nucleus to activate AOX expression is

unclear. One suggested mechanism is via generation of ROS. In tobacco suspension cells it

was found that upon addition of H2O2 within the span of two hours the level of AOX

mRNA was increased [67]. In S. guttatum it was found that application of salicylic acid led

to an increase in AOX mRNA levels [68]. AOX expression is also shown to be

developmentally regulated [69].

Post-translational regulation:

As mentioned previously, the plant AOX is a homodimer whereas the fungal AOX is a

monomer. The plant AOX is subject to two interrelated post-translational mechanisms of

regulation. In plants the alternative oxidase can be in an oxidised or a reduced form [61].

Plant AOX can be activated by reduction of a dimer-forming disulphide bridge. The

reduced (active) form is a non-covalently linked dimer whereas the oxidised (inactive) form

is covalently linked [70]. In transgenic tobacco plants expressing AOX it was found that

certain TCA intermediates (citrate, isocitrate and malate) could reduce AOX [71]. It was

hypothesized that the aforementioned intermediates may be involved in NADP reduction in

plants and that NADPH mediates reduction of plant AOX in vivo. This could be a means of

regulating AOX in response to changing matrix reduction levels. The second mechanism is

direct activation of AOX by certain organic acids. A study by Millar et al. [72] showed that

the plant AOX can be activated by a range of organic acids, most of them -keto acids:

pyruvate, hydroxypyruvate, glyoxylate, -ketoglutarate, oxaloacetate, L-malate and

succinate. It was determined that from these acids L-malate and succinate did not activate

AOX directly. It was found that in the absence of malic enzyme (in SMP’s) succinate and

malate could no longer activate AOX, which implies that it is in fact generation of pyruvate

via malic enzyme which causes activation. It was concluded that this type of plant AOX

activation is restricted to -keto acids. It was also determined that pyruvate activation is not

28

dependent on pyruvate metabolism which implies that pyruvate has a direct effect on AOX

[66]. As opposed to activation of AOX via pyruvate formation an alternative mechanism

was hypothesised by Wagner et al. [73] where addition of succinate or malate led to

changes in membrane fluidity which could facilitate the diffusion of QH2 from

dehydrogenase to AOX. A substrate dependent difference in AOX activity is commonly

seen where succinate dependent cyanide resistant respiratory rates are higher than NADH

ones [8, 73-76]. This could be explained by a change in membrane fluidity, however it is

more generally accepted that the higher cyanide resistant respiration rate with succinate is

due to production of endogenous pyruvate [77]. The activating effect of pyruvate on AOX

initially was assumed to change the affinity of AOX for QH2. In the absence of pyruvate,

plant AOX is known to activate only at relatively high levels of Q-reduction (between 35-

50% reduced) [78, 79]. Addition of pyruvate was seen to reduce the threshold level of Q-

pool reduction at which AOX becomes engaged [80] whilst pyruvate showed no effect on

the redox status of the AOX protein disulfide bond. The two mechanisms are interrelated

because pyruvate can only significantly activate AOX when the dimer is in the reduced

form [80]. Conversely, if pyruvate is present, significant AOX activation can only be

achieved when the dimer is reduced [81].

Interestingly enough many studies indicate that pyruvate can activate AOX activity with

succinate as a substrate [79, 80] although it has been reported that succinate itself can

activate AOX. This suggests that the amount of pyruvate generated indirectly from

succinate is not sufficient to fully activate AOX and that further addition of pyruvate is

required to fully activate AOX. It was concluded from experiments in which malic enzyme

was inhibited that differences in the generation of intramitochondrial pyruvate can explain

differences in AOX activity between tissues and substrates [77]. A pH effect on AOX

mediated respiration is seen in certain plant species [82], for instance with external NADH

as substrate S. guttatum mitochondria displayed a pH optimum for cyanide-resistant

respiration [83].

Activation of fungal and protist AOX is quite different from their plant counterpart.

AOX in fungi and protists are generally found as monomers and are not subject to organic

acid stimulation [61]. Interestingly, in recent work where the protist AOX from Ciona

29

intestinalis was expressed in human cells, pyruvate was found to activate the AOX protein

[84]. Purine nucleotides, such as AMP, GMP and IMP are reported to have an activating

effect on fungal and protist AOX [58]. Also an effect of pH on AOX activation in

Acanthamoeba castellanii mitochondria was found [82]. Despite differences between plant

and non-plant AOX at the level of regulation, monoclonal antibodies raised against

Sauromatum guttatum AOX cross-react with fungal and protist AOX proteins

Other factors regulating AOX are the amount of ubiquinone present [85], the Q-pool redox

poise [86] and the amount of AOX protein present [85, 87].

AOX Function:

The only function of AOX which has been commonly accepted is that of heat generation in

order to volatilise odiferous compounds in order to attract insects during pollination in

thermogenic plants [88]. Its function in non-thermogenic plants, let alone in non-plant

species to date is still a matter of debate. Several, not mutually exclusive, hypotheses have

been proposed. Continued turnover of the TCA cycle during any condition that inhibits or

decreases the activity of the cytochrome pathway, such as under ADP limited conditions or

during stress (wounding, chilling, drought etc.) will allow continuous production of

biosynthetic precursors [89]. Another possible function of AOX is to scavenge harmful

ROS produced under conditions (limited ADP, stress conditions) where components within

the respiratory chain become highly reduced [65]; by keeping the ETC relatively oxidised

AOX activity could prevent synthesis of ROS. Both hypotheses have in common that

inhibition of the cytochrome pathway leads to activation of AOX. A more recent hypothesis

suggests that AOX activity in plants serves as a means to keep plant growth relatively

stable under variable environmental conditions [90].

AOX in relationship to UCP:

Both AOX and UCP dissipate free energy as heat. It is interesting to note that some

organisms express both enzymes in their mitochondria [91, 92]. Affourtit et al. raise the

question as to what the physiological need could be for having two energy dissipating

30

enzymes in one system [37]. It has been demonstrated in Acanthamoeba castellanii

mitochondria that combined activity of both AOX and UCP leads to stronger reduction in

ROS formation than with either of the complexes being active alone [91]. Furthermore it

has been shown in plant mitochondria that activity of UCP appears to be coordinated with

AOX activity [92]. These observations suggest that when both energy dissipating

mechanisms are present in the same system their coordinated activities are involved in

reducing ROS concentration.

1.3 Schizosaccharomyces pombe

1.3.1 General background:

In this study S. pombe mitochondria were used as a model system to heterologously express

a plant alternative oxidase [59] in order to investigate its respiratory characteristics within

the respiratory chain. The same system has been used previously in our laboratory to

investigate structure-function relationships [26, 45, 46, 59, 63, 70, 93, 94]. Yeast systems

are a useful tool to investigate protein characteristics, it is relatively easy to express a

foreign gene and within a short time a large amount of the protein of interest can be

harvested. The yeast Schizosaccharomyces pombe also referred to as ‘the other yeast’ [95]

is increasingly the preferred model system to investigate a wide range of processes such as

the cell cycle [96], DNA repair [97], microtubule formation, meiotic differentiation,

cellular morphogenesis and stress response mechanisms [98] over the traditionally used

Saccharomyces cerevisiae (which recently has also been used to heterologously express a

plant alternative oxidase [30]).

S. pombe divides by fission and is one of the few free-living eukaryotic species

whose genome has been completely sequenced [99] at the time of writing. The S. pombe

genome is haploid and contains three chromosomes 13.8 Mb in size [98]. The

mitochondrial genome is 20 kb in size [99]. It has been reported that S. pombe resembles

mammalian cells more closely than does S. cerevisiae [100], for instance, S. pombe

recognizes various mammalian promoters, splices mammalian introns and shares the same

polyadenylation signals with mammalian cells, unlike S. cerevisiae [101]. It is also reported

31

that S. pombe genes have longer upstream regions on average than those of S. cerevisiae

which may mean that they are more complex and possibly more like those of higher

eukaryotes [98]. S. pombe has proportionally more genes conserved in metazoans than does

S. cerevisiae which is another argument in favour of the claim that S. pombe as an organism

is more closely related to higher eukaryotes than S. cerevisiae. On the other hand each yeast

shares genes with metazoans which the other lacks [102]. Furthermore a significant number

of chromosome associated proteins are absent in S. cerevisiae but shared between S. pombe

and metazoans making S. pombe the preferred system to study chromosome dynamics

[102]. In 2002 a total of 172 S. pombe proteins were found to have similarities to human

disease proteins whereas 182 such proteins were identified in S. cerevisiae. Most of the

genes coding for these proteins are shared between the two yeasts [99]. Therefore both

yeasts appear to be similarly useful as model organisms for the study of human disease

gene function although given their different biologies one organism could be preferred for

certain genes over the other and vice versa.

Although S. pombe has been extensively used to investigate the cell cycle and

genome repair mechanisms, in comparison to S. cerevisiae, relatively little work has been

done on S. pombe metabolism [103] and even less has been done on the respiratory

characteristics of its mitochondria [104, 105]. Recently however S. pombe mitochondria

have been used to investigate several bioenergetic processes. S. pombe is the preferred

system to investigate F1-ATPase (complex V) catalysis. The F1 part of complex V has

several nuclear encoded subunits (the and units). S. cerevisiae cannot produce mutants

of these subunits, leading to the production of “petite” colonies, i.e. cells with impeded

oxidative phosphorylation. Hence F1 mutants cannot be studied in S. cerevisiae [106]. It has

been a long held belief that S. pombe does not express a mitochondrial alcohol

dehydrogenase (ADH), recent work done in this laboratory however indicates otherwise

[26]. Mitochondrial ADH couples the oxidation of ethanol to the reduction of endogenous

NAD+ to NADH, which can subsequently be oxidised by an internal NADH

dehydrogenase, as happens in S. cerevisiae [107]. The presence of a gene encoding for

ADH in S. pombe was confirmed as far back as 1983 [108], it was concluded that the

protein was a cytosolic one [26]. The current hypothesis in this laboratory is that having

both a cytosolic and a mitochondrial ADH can function as a means to equilibrate

32

NAD+/NADH ratios on both sides of the IMM in a way similar to the situation in S.

cerevisiae [26]. The S. pombe gene SPAC5H10.06c was identified as a likely candidate

encoding the mitochondrial ADH isozyme. Because of a recent discovery of a homolog of

this protein in human liver [109] the discovery of a S. pombe mitochondrial ADH may have

potential medical implications [26].

Another typical protein involved in bioenergetic processes is the adenylate kinase

(AK) which catalyses the reaction: ATP + AMP 2 ADP [110]. In potato mitochondria

its activity was shown to be responsible for the relatively high respiratory rate under ADP

limited conditions [110]. Work done in this laboratory showed that continuous regeneration

of ADP (from either endogenous or added nucleotides) led to constant activity of the ATP

synthase affecting both membrane potential and oxygen consumption rate [38, 110, 111]. A

gene coding for AK in S. pombe has been identified [112] and results suggested that the

enzyme was found both in the cytosol and in the mitochondria.

1.3.2 The respiratory chain of S. pombe mitochondria:

The respiratory chain of S. pombe mitochondria is rather similar to the mammalian one,

see Figure 1.14:

Figure 1.14 Schematic representation of the S. pombe ETC. See legends of figures 1.3 and 1.4.

33

The S. pombe ETC, just like S. cerevisiae [27] does not contain complex I [13], therefore

the only means of generating a pmf is via the cytochrome pathway. Work done in this

laboratory [104] showed that isolated S. pombe mitochondria could respire on either

succinate or NADH (in a rotenone-insensitive manner) indicating the presence of complex

II and an external NADH dehydrogenase (which is nuclear encoded [113]) respectively.

The aforementioned findings on the S. pombe mitochondrial ADH indicate the presence of

an internal NADH dehydrogenase [26].

To the best of our knowledge the membrane potential across the IMM in isolated S.

pombe mitochondria had not been measured prior to this study, but results by Moore et al.

showed the occurrence of protein import into the matrix. This process could be abolished

by addition of valinomycin which confirmed the presence of a membrane potential [104].

Respiration in S. pombe mitochondria can be completely inhibited by cytochrome pathway

inhibitors which indicates the absence of an alternative oxidase3 [26, 104]. Unlike the yeast

Hansenula anomala (now Pichia anomala) [116] AOX expression cannot be induced in S.

pombe by incubation of the cells with antimycin A [59]. It has been proposed that cyanide

resistant respiration (due to the presence of AOX) is found only in non-fermentative and

Crabtree-negative yeasts (capable of fermentation but not under aerobic conditions) [117].

It was found, in general, that yeasts which do not display cyanide resistant respiration also

do not express complex I in their ETC [118]. Non-fermentative yeasts under conditions

where the cytochrome pathway is inhibited can still generate an electrochemical gradient of

protons via complex I using the alternative oxidase as a terminal oxidase. The pmf

generated could be utilised by complex V to drive the synthesis of ATP. It was

hypothesized by Veiga et al. [117] that non-fermentative yeasts express both complex I and

AOX as an alternative to cytochrome pathway respiration, whereas yeasts such as S.

cerevisiae and S. pombe (which do not express complex I [13]) use fermentation as an

alternative to cytochrome pathway respiration.

In section 1.2.5 it was mentioned that in S. cerevisiae mitochondria the Q-pool

displayed non-pool behaviour [27]. Given the similarities between the make up of the

3 Within older literature [114] but also in recent textbooks [115] (page 213) S. pombe mitochondria are

reported to display cyanide resistant respiration, which is incorrect.

34

respiratory chains of both yeasts this had implications for S. pombe. In this laboratory

experiments were done, employing the same techniques which were used in the S.

cerevisiae study and it was found that in S. pombe mitochondria the Q-pool does show pool

behaviour [26]. Also, it was found previously that S. pombe mitochondria display

antimycin resistant respiratory kinetics (see section 1.2.5) during NADH dependent

respiration, cf. Figure 2A in [119].

It was reported that in addition to the respiratory components described so far S.

pombe contains genes for several other respiratory linked proteins, namely: Gut2 encoding

a glycerol-3-phosphate dehydrogenase, hmt2 encoding a sulphide dehydrogenase and ura3

encoding a dihydroorotate dehydrogenase. All three proteins can donate electrons to the Q-

pool [26]. Upon performing a BLAST search4 (Basic Local Alignment Search Tool [120])

through the S. pombe genome another gene coding for a respiratory linked protein was

found. SPAC20G8.04c codes for an electron transfer flavoprotein-ubiquinone

oxidoreductase (ETF) which is a water-soluble matrix based complex that contains a FAD

moiety and can accept electrons from several dehydrogenases containing flavin [1].

In this study S. pombe is used to functionally express AOX [59]. The alternative

oxidase is non-protonmotive and its activity dissipates free energy. It was described in

section 1.2.11 that AOX and UCP have the capacity to act in synergism. The presence of

an uncoupling protein in yeast has been demonstrated [121]. It is therefore relevant to know

whether or not S. pombe expresses an uncoupling protein. It was found by Stuart et al.

[122] that the S. pombe genome did not contain any UCP homologs5. However, at the time

of that study (1999) the S. pombe genome was only partially sequenced (55%). At present

the whole S. pombe genome is known [99] and a recent BLAST search did not reveal any

UCP homologs.

1.3.3 SDH activation in S. pombe mitochondria:

Comparable to plant mitochondria, activation of SDH in yeast requires the addition of ATP

[123]. It is known that the mechanism of ATP activation is not due to unbinding of OAA

4 http://www.genedb.org/genedb/pombe/index.jsp

5 The same study also showed that the S. cerevisiae genome does not contain any UCP homologs.

35

[124]. And addition of ATP to S. pombe mitochondria only partially activates SDH. For full

activation the addition of glutamate (which leads to removal of OAA) is required [93]. Also

an inhibitory effect of the uncoupler CCCP (which leads to dissipation of the pmf) on

succinate dependent respiration in S. pombe mitochondria was found [26] in a way similar

to plant succinate dependent respiration [21].

1.4 Summary energy transducing systems

To recapitulate; energy transducing systems can be defined in terms of the chemiosmotic

theory put forward by Peter Mitchell [125].

An energy transducing system:

1) has a set of membrane located components which reversibly couples the

translocation of protons to oxidoreduction reactions which generates an

electrochemical potential of protons. (the ETC being an example of such a set of

components).

2) has a membrane located ATP hydrolysing proton pump which can work in reverse

driven by the aforementioned electrochemical potential of protons which leads to

the catalysis of the thermodynamically unfavourable reaction of ATP synthesis.

(complex V).

3) can use the aforementioned electrochemical potential of protons to directly or

indirectly drive the transport of substrates across the membrane. (e.g. succinate via the

dicarboxylate carrier [43] or ADP exchanged for ATP by the adenine nucleotide

carrier (ANC) [126]).

4) the systems of postulates 1,2 and 3 are located in a specialised coupling membrane

which has a low permeability to protons and to other ions in general. (e.g. the

IMM).

36

The chemiosmotic theory is generally accepted in the field of bioenergetics and it therefore

is considered paradigmatic in this work. Although generally accepted, as recently as 2005,

the theory is still questioned, see references [127-129].

1.5 A modular representation:

In order to study oxidative phosphorylation in this study we used a modular approach in

which ETC components are lumped into Q-pool reducing pathways and Q-pool oxidising

pathways [37], see Figure 1.15. The external NADH dehydrogenase and the combined

activity of both SDH and the dicarboxylate carrier are the reducing pathways. The

cytochrome pathway and the alternative pathway are the oxidising pathways. The Q-pool

can be viewed as a reservoir which can accept electrons from the reducing pathways and

can donate electrons to the oxidizing pathways. When all ubiquinone is reduced to

ubiquinol the reservoir is ‘full’ and when all ubiquinol is completely oxidized it is ‘empty’.

Under steady state conditions the rate with which electrons flow into the Q-pool equals the

rate with which they leave. The overall electron flux through the respiratory chain can then

be assessed by measuring the oxygen consumption rate. The steady state Q redox poise and

oxygen consumption rate values are dependent on the interplay between the activities of the

reducing and oxidising pathways. Activity of the cytochrome pathway leads to the

formation of a pmf, due to the backpressure of this proton gradient the activity of this

pathway can be limited. If now an uncoupler were added, the backpressure would be

relieved and the activity of the cytochrome pathway increases, which is reflected in an

increased steady state oxygen consumption rate with a concomitant oxidation of the Q pool.

Some reducing pathways are not fully active upon addition of substrate, e.g. SDH becomes

more active upon addition of ATP, which is reflected also in an increased steady state

oxygen consumption rate, but in this situation the Q pool would become more reduced.

Things are not as straightforward when a condition changes which affects reducing and

oxidising pathway activities simultaneously, as will be discussed in chapter 3. But for now

these examples illustrate clearly the general idea of the modular approach that is used in

this study and which has been used successfully in previous studies [76, 93, 130, 131].

This approach has two main tenets:

37

1) the Q-pool is homogenous, i.e. ubiquinone (ubiquinol) molecules can freely interact with

different dehydrogenases and oxidases.

2) Reducing pathways only interact with oxidising pathways via the Q-pool as an

intermediate, i.e. there is no direct interaction between these pathways.

Figure 1.15 A modular representation of the ETC with the Q-pool as the central intermediate between

pathways. Reducing pathways: external NADH dehydrogenase and the combined activities of SDH and the

dicarboxylate carrier. Oxidising pathways: the cytochrome pathway (complexes III, IV and cytochrome c) and

the alternative pathway consisting of the alternative oxidase only.

A different modular approach called ‘top-down’ metabolic control analysis using p as the

central intermediate has been successfully applied to both mammalian and plant

mitochondria [132, 133]. In that approach energy transducing processes are classified as

either p producers or p consumers. Three components are defined which communicate

with one another via p: the ‘respiratory chain’ (dicarboxylate carrier + ETC), the ‘proton

leak’ (proton leak, cation cycles etc.) and the ‘phosphorylating system’ (ATP synthase, the

phosphate carrier and the adenine nucleotide carrier) [132]. The approach used in this study

assumes the pmf to be constant [37].

38

1.6 Summary

Hopefully this introduction has managed to illustrate that mitochondria are a bit more than

just little cellular batteries, they are indeed very important in regulating cellular physiology.

Not only in plants or yeasts but as much in mammalian cells. Another trend of recent years,

it appears, is the realisation that mitochondria are more and more involved in many human

medical afflictions ranging from diabetes [134] to hearing disorders [135].

The alternative oxidase is quite often considered a typical plant protein and therefore

according to current funding body standards maybe not so ‘fashionable’ however given the

recent finding that AOX is also found in the animal kingdom and the fact that it has been

expressed in human cells [84] could place the alternative oxidase back in the picture as a

clinical tool for investigating human diseases.

1.7 Aims:

To set up a three electrode system which allows for simultaneous determination of oxygen

consumption rate (vO2), Q-redox poise (Qr/Qt) and membrane potential () in isolated

mitochondria (chapter 3). This was achieved and successful recordings were made.

SDH activation in potato mitochondria by ADP, ATP and oligomycin was hypothesized to

occur indirectly using as an intermediate. This was investigated (chapter 3) and it was

determined that SDH activation did not occur indirectly via .

S. pombe respiratory kinetics have previously been characterised in terms of oxygen

consumption rate and Q-redox poise under various energetic conditions, but to the best of

our knowledge the membrane potential had not yet been determined. A further

characterization of the S. pombe respiratory kinetics was undertaken (chapter 4). This

yielded some interesting oxidising pathway kinetics which have not been seen previously in

mitochondria from other species.

39

S. pombe has been used previously in this laboratory to heterologously express AOX. It

was found then that AOX expression led to a change in respiratory kinetics under various

energetic conditions. Kinetic curves were fitted to data obtained from malonate titrations

done on mitochondria respiring on succinate, which has yielded a limited amount of data

points. In this study a novel titration method (an NADH regenerating system) was used to

obtain a larger dataset and a possible effect of AOX expression on generation was

studied (chapter 5). This approach demonstrated that S. pombe mitochondria expressing

AOX display substrate dependent differences in oxidising pathway kinetics. Furthermore

an effect of on AOX activity could not be demonstrated.

40

Chapter 2

Materials and Methods

2.1 Isolation and purification of mitochondria

In this study mitochondria were isolated from (transformed) Schizosaccharomyces pombe

cultures, Saccharomyces cerevisiae cultures, fresh potato tubers and Arum maculatum

spadices.

2.1.1 Schizosaccharomyces pombe—The S. pombe strain used in this study is the so called

sp.011, ade6-704, leu1-32, ura4-D18,h- [104]. From this strain three types of yeast cultures

were grown, a wild-type (sp.011 wt) and two transformants. One type of transformant had

the S. guttatum AOX expressed, depending on the presence of thiamine in the growth

medium AOX was either expressed or repressed in the mitochondria (sp.011 AOX and

sp.011 AOX+T respectively). Another type of transformant had an empty vector expressed

(sp.011 pREP). Therefore a total of four different types of S. pombe mitochondria were

investigated in this study. All media used for yeast transformation, yeast growth and yeast

mitochondrial isolation are described in section 2.1.1.7.

2.1.1.1 The Expression system—Functional expression of a plant alternative oxidase in S.

pombe was achieved by using a system developed by Albury et al. [59]. A Sauromatum

guttatum cDNA clone (pAOSG81 [136]) which represents the nuclear gene aox1 [54] was

cloned into the expression vector pREP [137] in which it is under the control of the nmt1

promoter6. A transformed S. pombe culture will not express AOX when grown in the

presence of thiamine.

Apart from aox1 several other genes are present on the plasmid. The LEU2 gene (from S.

cerevisiae) codes for a protein involved in leucine biosynthesis. Using a S. pombe strain in

6 no message in thiamine

41

which the equivalent gene (leu1-) is disrupted the plasmid becomes essential for growth in a

medium lacking leucine. The ars1 gene (autonomously replicating sequence) is required for

initiation of replication in yeast, whereas the ori gene is required for initiation of replication

in bacteria. The AMP gene (resistance against amphicillin) provides a method to select for

the plasmid in bacteria. When expressed in S. pombe (grown in the absence of thiamine and

leucine) AOX is targeted to and incorporated into the IMM as a functional enzyme [59].

2.1.1.2 Yeast transformation—S. pombe cells were transformed with pREP-AOX (coding

for the S. guttatum AOX) or with pREP (just the vector) using a modified lithium acetate

method [101]. A single sp.011 wt colony was inoculated in 5 ml YES medium and

incubated overnight (150 rpm, 30 C) which normally grew to ~2.5x107 cells/ml. 800 l of

this culture was used to inoculate 100 ml of minimal medium, which was grown overnight

(150 rpm, 30 C).

The cells were harvested by bench-top centrifugation (2 minutes at 3000 rpm at room

temperature) and washed in distilled water. The cells were then resuspended in 0.1 M

lithium acetate / Tris-EDTA (LiA/TE) in 10 0.1 ml aliquots at a density of ~1x109 cells/ml.

The cells were then incubated at 30 C (waterbath) for one hour with occasional mixing. To

each aliquot 1-2 g DNA (in ~10 l) and 290 l 50% polyethylene glycol (PEG) dissolved

in LiA/TE was added. The cells were then again incubated for one hour in the waterbath at

30 C with occasional mixing. This was followed by a heat shock step of 15 minutes at 43

C (waterbath) for 15 minutes. Cells were then pulsed to a pellet and the supernatant

removed. The pellet was gently resuspended in 100 l 0.1 M LiA/TE. The cells were then

plated on minimal medium agar plates and grown for 3-5 days at 30 C. Colonies grown on

these plates were subsequently used to inoculate larger cultures.

2.1.1.3 S. pombe growth—A starter culture was set up by picking a colony from a yeast

plate and adding it to a 200 ml solution of minimal media. Supplements were added

depending on yeast type. 0.4 mM adenine and 0.7 mM uracil were added to all cultures. In

the case of sp.011 AOX+T 0.5 mM thiamine was added to repress expression of AOX. The

wild type sp.011 WT requires addition of 1.1 mM leucine. A culture was incubated under

42

aerobic conditions for three days at 30 C (150 rpm) for the cells to reach stationary phase.

Cell concentration was assessed spectrophotometrically (light scattering at A595 [93]) using

a 15-fold dilution of the cell culture in distilled water. This value was used to calculate the

volume of culture required to inoculate each of four fresh 1 litre culture flasks (minimal

medium with the appropriate supplements) to give cell concentrations ~ 40% of the

stationary phase density at the anticipated time of harvesting. For this the following

exponential growth equation was used:

[2.1]

Where is the growth rate, N0 is the starting concentration, Nx the final concentration and

t the period of growth. After rearranging this gives:

[2.2]

In this form N0 represents the starting cell density which gives the required Nx density after

t time (hours) with cells growing at a rate of . In a previous study values of of ~0.14

and 0.12 h-1

for non-transformed and transformed cells were determined [26]. In this study

similar values were obtained. The inoculated 1 litre flasks were typically incubated for 19-

21 hours under aerobic conditions at 30 C and 150 rpm.

t

NN x

)log(log 303.2 0

)303.2

(log

0 10

tNx

N

43

2.1.1.4 Isolation of mitochondria from S. pombe cultures—On the day before isolation four

1 litre flasks were sub-cultured by adding a certain volume of starter culture, the amount of

which calculated as described in 2.1.1.3. The isolation of mitochondria from S. pombe

cultures can be broadly divided into two stages. 1: Spinning down of the yeast cells from

the four 1 litre cultures and treatment of the cells with lysing enzymes to remove the outer

membranes and induce spheroplast formation. 2: Inducing spheroplast lysis by osmotic

shock and subsequent harvesting of the mitochondria through differential centrifugation.

The isolation protocol used here is based on the method of [104].

2.1.1.5 Spheroplast preparation—Cells were harvested by centrifugation (10 minutes, 7000

rpm). Cells were washed by resuspension in distilled water at 4 C and again spun down

(10 minutes, 7000 rpm). The wet weight was recorded (typically between 15-20 g). Cells

were resuspended in 200 ml spheroplast buffer (SB) and incubated at 30 C, 150 rpm for 15

minutes in the presence of the cell wall-digesting enzyme preparation Zymolyase 20T7 (5

mg / g wet weight). Upon addition of a second preparation, ‘lysing enzyme’8 (15 mg / g wet

weight) the suspension was incubated for a further 45 minutes at 30 C, 150 rpm.

Spheroplast formation was subsequently assessed spectrophotometrically by diluting a

suspension aliquot 100x in distilled water. The level of scattering (A800) due to cells and

intact spheroplasts compared to cells not treated with digestive enzymes was used to

indicate the proportion of osmotically sensitive spheroplasts in the sample. Also, a 5 l

aliquot of cell suspension was osmotically shocked by addition of 5 l distilled water, the

effect of which was observed using a light microscope. Due to removal of the cell wall this

led to lysis.

2.1.1.6 Isolation of mitochondria—All procedures described from here on were performed

on ice to minimize enzymatic activity. The spheroplast suspensions were diluted 2-fold in

spheroplast wash (SW) and spun down for 10 minutes at 1600 rpm at 4 C. As a washing

step the pellets were resuspended in SW and again spun down for 10 minutes at 1600 rpm

at 4 C. Pellets were resuspended in ~2 ml mannitol wash (MW) and transferred to a glass

7 Seikagaku corporation, code number: 120491

8 Sigma, code number: L1412

44

homogeniser. With two gentle strokes the resuspended pellets were homogenised, the total

volume was subsequently increased to 600 ml with MW to lyse the spheroplasts (osmotic

shock). Cell debris and unlysed cells were removed by centrifugating at 3500 rpm for 15

minutes at 4 C. The supernatant was subsequently centrifuged at 13000 rpm for 10

minutes at 4 C. Pellets highly enriched with mitochondria were pooled and centrifuged at

10000 rpm for 10 minutes at 4 C to yield a final mitochondrial pellet which was

resuspended in a small volume of ~1-2 ml of MW and kept on ice throughout the remainder

of the experimental day.

2.1.1.7 S. pombe media—The following media were used for the transformation of S.

pombe cells, the growth of S. pombe cultures and the isolation of mitochondria from these

cultures.

YES medium (Yeast Extract with Supplements):

amt component final conc

5 g/l yeast extract 0.5% w/v

30 g/l glucose 3.0% w/v

Supplements: 225 mg/l adenine, histidine, leucine, uracil and lysine hydrochloride.

Solid media (for plates) was made by adding 2% Difco Bacto Agar.

Transformation media:

0.1 M LiA/TE : 0.1 M lithium acetate in Tris-HCl (10 mM, pH 7.6) and EDTA (1 mM).

50% PEG in 0.1 M LiA/TE made up fresh on the day of transformation, not sterilised.

45

Minimal medium [138]:

0.11 M glucose 19 µM FeCl3.6H2O

93 mM NH4Cl 0.9 µM Na2MoO4.H2O

15 mM Na2HPO4 0.6 µM KI

15 mM KH-Phthalate 0.2 µM CuSO4.5H2O

5.2 mM MgCl2.6H2O 5 µM citric acid

0.1 mM CaCl2 1 µM Na pantothenate

14 mM KCl 80 µM nicotinic acid

0.3 mM Na2SO4 55 µM inositol

8.0 µM H3BO3 40 nM biotin

1.8 µM MnSO4.4H2O 1 mM NaOH

1.4 µM ZnSO4.7H2O

Solid media (for plates) was made by adding 2% Difco Bacto Agar.

Spheroplast buffer (pH 5.8)

1.35 M sorbitol

1 mM EGTA

10 mM Citrate/phosphate

Citrate/phosphate: 100 mM citric acid and 100 mM Na2HPO4 pH 5.8 mixed in ratio

~50:200

Spheroplast wash (pH 6.8)

0.75 M sorbitol

0.4 M mannitol

10 mM MOPS

46

Mannitol wash (pH 6.8)

0.65 M mannitol

2 mM EGTA

10 mM MOPS

Yeast reaction medium (pH 6.8)

0.65 M mannitol

1 mM MgCl2

5 mM Na2HPO4

10 mM NaCl

20 mM MOPS

2.1.2 Saccharomyces cerevisiae—The S. cerevisiae strain used in this study was ordinary

baker’s yeast ‘Carrs – breadmaker yeast’ purchased at a local supermarket.

A small quantity of dried yeast was dissolved in distilled water and subsequently plated on

YES medium (section 2.1.1.7) based agar plates to grow S. cerevisiae colonies.

The same protocols used for growing S. pombe cultures and isolating S. pombe

mitochondria were used with S. cerevisiae with some minor alterations. The starter culture

was grown for one day only, as opposed to three days and in the degradation step only

Zymolyase was used (5 mg / g wet weight), the lysing enzymes were omitted. For

electrochemical experiments the yeast reaction medium was used (section 2.1.1.7).

2.1.3 Potato tuber—Fresh potato tubers were bought at a local supermarket. The protocol

to isolate and purify mitochondria from this tissue is based on [139]. Media used in this

protocol are described in section 2.1.3.2. All operations were performed on ice to minimize

enzymatic activity.

47

2.1.3.1 Isolation of mitochondria from potato tubers—Potatoes (~1.5 kg) were peeled

thickly, cut into large chip-sized pieces and homogenised in grinding medium using a juice

extractor (Moulinex type 140). The juice was collected directly in 1.3 liter of grinding

medium (GM). The homogenate was then filtered through a moistened muslin (to remove

large starch particles). The pH was adjusted to 7.4. Subsequently, three centrifugation steps

were performed, all at 4 C. First the homogenate was centrifugated for 5 minutes at 1500

rpm (to completely get rid of starch). The supernatant was centrifugated for 10 minutes at

4000 rpm. The supernatant was then centrifugated for 15 minutes at 10000 rpm. The pellet

was resuspended in 2-5 ml washing medium (WM). The suspension was transferred to two

50 ml centrifuge tubes and WM was added to fill the tubes. This was followed by another

centrifugation step of 10000 rpm for 10 minutes at 4 C. The pellets were resuspended in a

small volume (2-5 ml) of WM and pipetted on top of a self-forming PercollTM

gradient in

25 ml of purification medium (PM). The tubes were centrifugated at 18000 rpm for 30

minutes at 4 C. Using a pastette the mitochondria were removed from the gradient and

diluted in WM (at least 1:10). Purified mitochondria were pelleted by a centrifugation step

of 10000 rpm for 10 minutes at 4 C. The mitochondrial pellet was then resuspended in a

small amount of WM (1-2 ml) and kept on ice for the remainder of the experimental day.

2.1.3.2 Potato tuber media:

Grinding medium (pH 7.4)

0.3 M mannitol

0.1% w/v BSA

40 mM MOPS

2 mM EDTA

0.6% w/v PVP 40

10 mM cysteine

48

Washing medium (pH 7.4)

Identical to grinding medium apart from the fact that cysteine is omitted.

Purification medium (pH 7.4)

21% v/v PercollTM

0.3 M sucrose

5 mM MOPS

0.1% w/v BSA

Potato reaction medium (pH 7.2)

0.3 M mannitol

1 mM MgCl2

5 mM K2HPO4

10 mM KCl

20 mM MOPS

2.1.4 Arum maculatum—The protocol to isolate and purify mitochondria from this tissue

was based on [88]. Media used in this protocol are described in section 2.1.4.2. All

operations were performed on ice to minimize enzymatic activity.

2.1.4.1 Isolation of mitochondria from Arum maculatum spadices—Spadices from local

Sussex woods were isolated from the leaf tissue and chopped into small ~1 cm3 slices and

added to ice-cold grinding medium (GM). The slices were homogenised in a WaringTM

blender in 2x3 s bursts. The homogenate was filtered through a wetted muslin and

centrifugated at 4000 rpm for 10 minutes at 4 C. The supernatant was then centrifugated at

10000 rpm for 10 minutes at 4 C. The pellet was resuspended in washing medium (WM)

which was then centrifugated at 12000 rpm for 10 minutes at 4 C. The pellet was

resuspended in a minimal amount of WM and loaded onto a 21% PercollTM

self-forming

49

gradient. The gradient was centrifugated at 14000 rpm for 30 minutes at 4 C. The

mitochondrial band was removed using a pastette and transferred to WM. The

mitochondrial suspension was then centrifugated at 12000 rpm for 10 minutes at 4 C.

Mitochondria were gently resuspended in a small volume (1-2 ml) of WM and kept on ice

during the remainder of the experimental day. Mitochondria were isolated either

immediately after picking of the spadices or after storing the spadices overnight at 4 C.

2.1.4.2 Arum maculatum media:

Grinding medium (pH 7.5)

0.3 M mannitol

0.2% w/v BSA

20 mM MOPS

2 mM EDTA

2 mM pyruvate

7 mM cysteine

Washing medium (pH 7.5)

Identical to grinding medium apart from the fact that cysteine is omitted.

Purification medium (pH 7.5)

21% v/v PercollTM

0.3 M sucrose

5 mM MOPS

0.1% w/v BSA

2 mM pyruvate

Reaction medium

The same as for potato, see section 2.1.3.2.

50

2.1.5 Specifics of plant mitochondrial isolation [38, 140]:

Plant cells have a rigid cell wall which requires the use of shearing forces to disrupt, in

order to liberate cytoplasmic organelles. This leads inevitably to the rupture of the cell

vacuole thereby releasing harmful compounds, such as hydrolytic enzymes, phenolic

compounds, tannins, alkaloids and terpenes, which can interact with the mitochondrial

membranes. In order to minimise interaction of these compounds with the mitochondria

several precautionary measures can be taken. Phenolic compounds and their oxidation

products (quinones) being highly reactive can react strongly with mitochondrial

membranes. Bovine serum albumin (BSA) is routinely used, not only to bind free fatty

acids, but also because it can bind to quinones. Cysteine which is added to the grinding

medium of potato and arum preparations is another protective agent preventing quinone

interactions. Polyvinylpyrrolidone acts as a scavenger of phenols and tannins.

pH is kept between 7.2-7.5 as alkaline pH will increase phenol autooxidation and acid pH

will increase interaction between phenols and protein functional groups.

2.2 Polyacrylamide gel electrophoresis & Western analysis

2.2.1 SDS-PAGE—Proteins were separated using 1-D SDS PAGE. Mitochondrial samples

(stored at –80 C) were defrosted on the day of electrophoresis. Mitochondrial protein (15

µg per lane) was separated on 0.75 mm thick 10% SDS-polyacrylamide gels according to

the method of [141]. Electrophoresis was performed for ~ 1 hour at 150 V. Samples were

run under non-reducing conditions through the omission of -mercaptoethanol in the gel.

2.2.2 Blotting to nitrocellulose—Separated proteins were transferred from SDS-

polyacrylamide gels to nitrocellulose membranes using standard electrophoretic methods

[142]. Transfer was carried out in ice cold transfer buffer (25 mM Tris-192 mM glycine,

10% v/v methanol, pH 8.8) for 1 hour at 100 V.

51

2.2.3 Immuno-detection of proteins—After blotting, nitrocellulose membranes were washed

in Tris-buffered saline (TBS; 140 mM NaCl, 20 mM Tris-HCl pH 7.6) and gently agitated

overnight at 4 °C in blocking solution (BS; 2% w/v milk powder 3% w/v BSA and 0.1%

v/v Tween 20 in TBS). This was followed by 6 x ~ 5 min washes in TBS, filters were

incubated for 1 hour at room temperature in BS containing mouse monoclonal, anti-AOX

(from Sauromatum guttatum) antibodies [53] (1:2000 dilution). Filters were washed in

TBS, as before, and then incubated for 1 hour in BS containing a 1:1000 dilution of

secondary antibody (linked to horseradish peroxidase). Antibodies were detected on light-

sensitive film using an enhanced chemiluminescence kit (Amersham International plc). S.

guttatum AOX antibodies were a gift from Dr. Tom Elthon (University of Nebraska).

2.3 Protein estimations

Due to the time consuming nature of the isolation of mitochondria and subsequent

experiments; protein estimations were normally done on a different day. Therefore isolated

mitochondria were kept frozen at –80 C and defrosted on the day of protein estimation.

Protein concentrations were determined using a bicinchoninic acid (BCA) assay [143] in

the form of a kit (BCA, Pierce, Rockford, UK) with bovine serum albumin (BSA) as a

standard. In this assay, a solution of protein is incubated with a solution containing cupric

sulfate and BCA. The cupric ion (Cu2+

) is reduced to the cuprous ion (Cu+) by proteins in

an alkaline medium. This reaction is often referred to as the Biuret reaction. The cuprous

ion then forms a purple-colored complex with BCA that strongly absorbs light at 562 nm.

All samples were kept for 30 minutes at 37 C before determining absorbances in a

spectrophotometer (CARY 400 Scan). A calibration curve (absorption vs. mg protein) was

made using a series of BSA standards by diluting a stock solution of BSA of 2 mg/ml with

distilled water, including one cuvette filled with distilled water as a blank. This was done in

duplicate and the absorption values were measured in a spectrophotometer. The acquired

values were plotted in Kaleidagraph (version 3.02) and fitted using a linear fit. The

mitochondrial samples with unknown protein weight were diluted 20 and 50 times (both in

duplicate) and the acquired absorbance values were inserted into the equation which was

derived from fitting the calibration curve (also done in Kaleidagraph).

52

2.4 Electrochemical techniques

All electrochemical experiments were performed in a setup as shown in Figure 2.1. A

perspex reaction chamber was constructed by the University of Sussex mechanical

workshop which could accommodate a volume of 2.2 ml (C). After cleaning the vessel with

pure ethanol9 and rinsing with distilled water, reaction medium was added. The chamber

sits atop a magnetic stirrer (A) which drives a stirring bean present in the chamber to keep

the reaction medium homogenous. The vessel can be airtight sealed with a stopper (D)

which contains a 1 mm channel through which chemicals can be added via a micro syringe.

An oxygen electrode (described in 2.4.2) is mounted in the bottom of the reaction chamber.

On the sides of the chamber a total of 4 plugs are present which can be removed in order to

add more electrodes to the setup (the Q-electrode and the TPP+ electrode).

Figure 2.1 The reaction chamber used in this study. A: magnetic stirrer. B: oxygen electrode.

C: reaction chamber. D: stopper.

9 With yeast mitochondria propan-2-ol was used as ethanol is a substrate for these mitochondria. With all

other tissues ethanol was used to clean the vessel.

A

D

C

B

53

2.4.1 The oxygen electrode—The concentration of dissolved oxygen was measured using a

Rank oxygen electrode [144]. The electrode consists of a Ag/AgCl reference anode and a

platinum cathode, see Figure 2.2. The electrodes are immersed in a saturated KCL solution

and separated from the reaction medium by an oxygen-permeable Teflon

membrane. A

potential difference of ~0.7 V is set up between the anode and the cathode using a

potentiostat (Rank Brothers Ltd. Bottisham, Cambridge UK).

The following reactions occur at the electrodes:

4 Ag + 4 Cl- 4 AgCl + 4 e

- (anode)

4 H+ + 4 e

- + O2 2 H2O (cathode)

At the platinum electrode electrons reduce oxygen molecules to water whilst chloride ions

migrate to the anode where they release electrons. The overall result is a transfer of

electrons from anode to cathode causing a current flow between the two electrodes which is

proportional to the concentration of oxygen in the reaction chamber. As oxygen is

consumed in this process it can be appreciated that oxygen concentration near the

electrodes will decrease over time therefore it is necessary to have a stirring bean present to

keep the solution homogenous.

As the current between the electrodes is proportional to the concentration of oxygen and the

response is linear only two calibration points are necessary [144]. Air saturated reaction

medium gives the signal corresponding to maximum oxygen concentration in aqueous

solution (250 M at 25C [145]) and sample-induced anaerobiosis gives the signal

representing zero oxygen. A more conventional way of achieving anaerobiosis through the

addition of sodium dithionite (Na2S2O4) is not used to calibrate the oxygen electrode in this

study as it severely affects the functioning of the Q-electrode.

54

Figure 2.2 Oxygen electrode mounted in the bottom of the reaction chamber (see Figure 2.1).

Platinum electrode in the middle is the cathode, the annular silver electrode the anode.

2.4.2 The Q-electrode—The Q-electrode allows for the continuous measurement of the Q-

redox poise in mitochondria and has been employed successfully in isolated mitochondria

from plant [21, 22, 76, 85, 86, 146-149], yeast [70, 93, 104] and mammalian systems [21].

The Q-electrode has also been used to determine the redox state of the plastoquinone pool

in thylakoids [150] and the redox state of the ubiquinone pool in bacterial membrane

fragments [151]. This apparatus consists of glassy carbon, platinum and Ag/AgCl

electrodes mounted in a sample vessel which allows amperometric measurement of the

redox poise of the endogenous Q-pool of isolated mitochondria (devised by Prof. Peter

Rich, Imperial College, London; see European patent No. 85900699.1 for details [152]).

Endogenous Q10 is water-insoluble and therefore bound to the IMM where it cannot be

detected by an electrode. Measurements therefore rely on a Q-mimic, Q1 (2,3-dimethoxy-5-

methyl-6-isoprenyl-p-benzoquinone) that is sufficiently soluble in both membrane and

aqueous environments to allow redox-equilibration with Q10 in the membrane as well as

55

interaction with an electrode surface in aqueous solution to communicate the relative poise.

Although the exact nature of Q1:Q10 redox-equilibration is unclear, it has been suggested

that it involves Q10H2:Q1 transhydrogenase activity at the Qn site of the mitochondrial bc1-

complex (P.G. Crichton D. Phil thesis University of Sussex [26] [Prof. Peter Rich,

University College London, personal communication]). The advantage of using this

technique is that it allows continuous measurements of the Q-redox poise whilst

simultaneously recording the oxygen consumption rate. Another technique which is used at

times is the determination of the Q-redox poise via Q extraction [76, 82] where

mitochondria in steady state are chemically quenched and subsequently have their quinones

extracted. Analysis of redox state is then determined using HPLC. Experiments where both

techniques were used on the same mitochondrial preparation gave similar results

corroborating the notion that Q-redox poise determinations obtained via the Q-electrode are

accurate [22, 76].

Due to limitations in the way that electron transfer occurs between quinones and

electrode surfaces (for mechanistic reasons the reaction is electrochemically irreversible

[152]), the redox poise of Q1 cannot be measured potentiometrically. Instead, the relative

concentration of Q1 or Q1H2 specifically, is measured using an amperometric method. In

the electrical setup (see Figure 2.3) an external voltage is applied to poise and maintain a

glassy carbon working electrode at –360 mV with respect to a Ag/AgCl reference electrode.

This is achieved using a potentiostat (University of Sussex Workshops) where a third

‘auxiliary’ electrode (platinum) along with the working electrode accepts the current in

order to avoid polarisation of the reference electrode (which would otherwise result in a

potential that varies with current).

The combination of electrodes constituting the Q-electrode is typically introduced

into an oxygen electrode chamber to allow simultaneous determination of Q-redox poise

and oxygen consumption (see Figure 2.4). Importantly, the measured current that flows

between the auxiliary and working electrode is proportional to the Q1 concentration.

Changes in the Q10 redox poise, and consequently the Q1 concentration, will therefore be

reflected in a change in the measured current. During respiratory measurements the system

is calibrated by measuring the signal associated with mitochondria in the absence of

respiratory substrates where the endogenous pool of Q10 is assumed to be fully oxidised.

56

The signal gained following sample-induced anaerobiosis is used to represent a fully

reduced Q-pool. The experimental values relative to these parameters can then be

estimated.

Figure 2.3 Schematic representation of the Q-electrode as adapted from [153].

Figure 2.4 Q-electrode combined with oxygen electrode. Working electrode (glassy carbon) blue wire.

Auxiliary electrode (platinum) red wire. Reference electrode (Ag/AgCl) green wire (at the back).

57

Several substances have been found to interfere with the Q-electrode:

NADH, malonate, TMPD, ascorbate, octyl gallate and ammonium sulfate. Precautions have

to be taken when using these substances during experiments.

2.4.3 The TPP+-electrode—In this study a tetraphenyl phosphonium (TPP

+) electrode was

constructed based on the method of [154]. The TPP+ electrode is an ion-selective electrode

[155]. The membrane potential across the IMM is negative inside with respect to the

outside, therefore a positive lipophilic ion must be used. In this study the selected ion was

TPP+ (see Figure 2.5) a lipophilic cation which can readily permeate the IMM. Due to

extensive -orbital systems the charge of the TPP+ ion is delocalised. Therefore TPP

+ ions

can move freely in both aqueous and hydrophobic environments [1]. Other ions which have

been used with ion-selective electrodes are triphenyl-methylphosphonium (TPMP+) [110]

and dibenzyl dimethyl ammonium (DDA+) in combination with tetraphenyl boron (TPB

-)

[156]. TPP+ has some advantages over other ions, e.g. it permeates the IMM 15 times faster

than DDA+ [154]. Some ions such as DDA

+ require the presence of a lipid-soluble anion

such as tetraphenyl boron (TPB-) for permeation through the IMM [154] whereas TPP

+

does not require any other lipid soluble ions to be present in order to permeate the IMM.

Figure 2.5 Structure of tetraphenyl phosphonium.

Due to the ease with which lipophilic ions such as TPP+ can permeate through the IMM in

response to changes in they have been successfully employed as indicator ions to

determine the magnitude of [155]. Ion selective electrodes do not measure the

directly. Instead the TPP+ electrode measures the concentration of TPP

+ ions in the

medium. Upon energization of mitochondria, TPP+ ions in the medium start migrating into

the matrix, attracted by the negative electrical potential on the matrix side of the IMM. This

58

results in a decrease in TPP+ ions outside of the mitochondria, [TPP

+o] and an increase of

matrix TPP+ ions, [TPP

+i]. By using the Nernst equation one can calculate from the steady

state [TPP+

i] and [TPP+

o] the value of the across the IMM:

][

][log

zF

RT2.303(mV)

i

o

TPP

TPP [2.3]

At room temperature this equation simplifies to:

][

][log95(mV)

i

o

TPP

TPP [2.4]

This means that for every tenfold difference in the TPP+

concentration ratio across the IMM

an electrical potential difference of 59 mV will be detected. Or alternatively, for every 59

mV increase in a tenfold accumulation of TPP+

i occurs. Given the ionic nature of the

TPP+ molecules, migration of these ions across the IMM could possibly lead to a change in

. To avoid a possible uncoupling effect (as TPP+ is a positive ion) a low concentration

of TPP+ was chosen to work with in this study. A TPP

+ concentration of 1 M was used.

Comparable studies where the TPP+-electrode was employed report values for TPP

+

concentration up to 10 M without any effect on [154, 157, 158]. Addition of TPP+ up

to 2.5 M did not have an effect on the respiration rate in the mitochondria used in this

study, therefore no uncoupling was induced through the addition of TPP+.

In order to determine [TPP+

i] a conversion factor was used where the matrix volume

was deduced from its protein content according to [159] with a value of 2 l mg-1

for potato

mitochondria and with a value of 1.02 l mg-1

for yeast mitochondria [160].

Membrane potentials were calculated using equation 2.4. The TPP+-electrode can only

determine the concentration of TPP+ in the medium (i.e. any TPP

+ molecules present in the

matrix cannot be detected). From [TPP+

o] the amount of TPP+ molecules in the medium can

59

be calculated. Combined with the total amount of TPP+

molecules added (2 nmole) [TPP+

i]

can be calculated:

[2.5]

2.4.3.1 The TPP+-electrode setup—Figure 2.6 shows the TPP

+-electrode setup.

The TPP+-electrode consists of a chlorated silver wire which is inserted into a PVC tube

(see Figure 2.7) filled with a solution of TPP+ (10 mM) dissolved in distilled water. A

Ag/AgCl reference electrode makes contact with the same solution as the TPP+-electrode.

The difference in potential between these two electrodes is detected by a Voltmeter

(University of Sussex Workshops).

Figure 2.6 TPP+-electrode setup. The TPP

+-electrode is inserted into the reaction chamber on the side.

In this configuration and oxygen consumption rate can be determined simultaneously.

umematrix vol

TPPTPPTPP mediumtotal

i

][

60

2.4.3.2 Detection of [TPP+

o]:

Detection of TPP+ ions in the medium by the TPP

+ electrode is based on ion exchange.

As mentioned previously a chlorated silver wire is inserted into a PVC tube which is sealed

on one end by a membrane that acts as a surface for ion exchange on both sides. The

membrane is faced with two solutions, the medium in the reaction chamber with a variable

concentration of TPP+

(depending on mitochondrial energy status) and an internal solution

of fixed TPP+ concentration. Before inserting the silver wire, the PVC tube is filled with a

10 mM TPP+ solution. The membrane is made out of PVC with tetraphenyl boron (TPB,

Na+

salt) imbedded as an ion exchanger (construction of the membrane will be discussed in

the next section). The following ion exchange occurs:

TPB-Na (membrane) + TPP+ (solution) = TPB-TPP (membrane) + Na

+ (solution)

Formation of the TPB-TPP complex is called conditioning (discussed in 2.4.3.4). After

conditioning the membrane presents a surface of TPP+ to both the internal solution and the

medium. As there is TPP+ in solution and on the membrane surface a boundary potential

will form due to activity (concentration) difference between membrane surface and

solution. That means there are two boundary potentials, the inner boundary potential is

constant since the internal TPP+ concentration is also constant, this forms a stable

reference. On the outer surface the boundary potential will be a function of the TPP+

concentration in the medium. The overall potential of the electrode will therefore be

governed by the concentration of TPP+ in the medium.

2.4.3.3 Construction of the TPP+-electrode membrane:

A 3 ml 10-2

M Na-tetraphenylboron (TPB) solution (dissolved in tetrahydrofuran(THF))

was mixed with a 10 ml solution of 0.5 g polyvinylchloride (PVC) dissolved in THF. This

solution was mixed with 1.5 ml of dioctylphtalate (a plasticiser). The solution was poured

into a fat free glass Petri dish (60 cm2) and was left evaporating overnight in a flowhood

avoiding any contamination with dust particles.

61

Construction of the PVC sleeve:

PVC tubing 5 mm outer diameter and 3 mm inner diameter was used. With a heated scalpel

a length of about 4-5 cm was cut off making sure the cut-off area had a smooth surface.

Construction of the TPP+-electrode:

A circle of the same outer diameter as the PVC tubing was stamped out of the membrane

and positioned on one of the ends of a 4-5 cm long PVC sleeve. The end to which the

membrane was attached was dipped in THF in order to ‘glue’ the membrane to the PVC

sleeve. The sleeve with glued on membrane was left to dry overnight. Then the sleeve was

for ¾ filled with a solution of 10-2

M TPPCl (in distilled water).

The silver wire was chlorated in a bath of 0.1 M HCl in which the silver wire and a

platinum wire were inserted. Both wires were connected to an electrical circuit over which

a potential of 50 mV was imposed. Electron flow through this circuit was from the silver

wire to the platinum wire. A current of 50 mA for the duration of 3 minutes was enough for

proper ‘whitening’ of the silver wire, i.e. homogenous depositing of AgCl. After rinsing

with distilled water the wire was carefully inserted into the sleeve. Figure 2.7 shows the

components of the TPP+-electrode.

2.4.3.4 Conditioning of the TPP+-electrode:

TPP+ electrodes sleeves are not instantly usable after the membrane is glued to it.

After several hours of drying (evaporation of THF) the PVC sleeve was filled with a 10

mM TPP+

solution. The sleeve was soaked in a solution of the same concentration for

several hours during which ion exchange could occur on both sides of the membrane.

62

Figure 2.7 Components of the TPP+-electrode setup. A: Ag/AgCl reference electrode (same type is used with

the Q-electrode) B: PVC TPP+-electrode sleeve C: AgCl coated silver wire. See text for details.

2.4.3.5 Calibrating the TPP+-electrode:

Potential differences between the TPP+ electrode and the reference electrode were recorded

using a MacLab/200 (Macintosh Operating Systems, AD Instruments Pty Ltd) and chart

analysis software (Chart v3.6/s, AD Instruments Pty Ltd) on an iMac desktop computer. As

explained in section 2.4.3 the TPP+-electrode does not measure the membrane potential

directly and although the recorded signals are in units of Volt they do not bear a direct

relationship to the membrane potential across the IMM. The TPP+-electrode detects

changes in TPP+ concentration in the medium and therefore the recorded signals in V need

to be related to concentration of TPP+. In order to do this; at the beginning of each

experiment a calibration trace is made, see Figure 2.8.

63

Figure 2.8 Calibration of the TPP+-electrode. The arrows indicate additions of TPP

+ to the medium (prior to

addition of mitochondria) which leads to downward deflections of the signal. A final concentration of 1 M is

reached, equal amounts of TPP+ (0.25 M) are added.

2.4.3.6 TPP+-electrode correction:

No correction for binding was used. During anoxia, upon addition of CCCP (or

valinomycin) the TPP+ signal went back to its original value (before mitochondria were

added) indicating that no significant binding had occurred.

2.4.3.7 TPP+-electrode sensitivity:

Upon calibration it was found that the TPP+-electrode had Nernstian slope values of 59 mV

at concentrations of TPP+

as low as 0.25 M. Throughout the experiments in this study a

concentration of 1 M was used. In the literature it is reported that the TPP+-electrode

responds linearly with a TPP+ concentration of 0.5 M and upwards [154]. It has been

reported that at TPP+ concentrations greater than 5-10 M respiration is inhibited [161].

64

2.4.3.8 Durability of TPP+-electrodes:

TPP+-electrodes (in this context the PVC sleeves with glued on TPP

+ membrane) had a

variable durability. One electrode lasted for months whereas some electrodes had to be

thrown away after only one or two experiments. There are two factors which indicate that

an electrode needs to be replaced:

- Decrease in response time

- Upon calibration the slope of the relationship between electrode deflection signal and

concentration of TPP+ in the medium no longer has a Nernstian value (i.e. a value

different from 59 is determined)

The approach taken in this thesis work was to make several TPP+-electrodes at a time and

to dispense of an electrode at the first sign of trouble, as opposed to trying to repair leaky

electrodes by resealing the membrane to the sleeve with THF. Throughout this thesis work

between 15 to 20 electrodes were used.

2.4.3.9 TPP+-electrode response time:

It can be seen from Figure 2.8 that upon addition of TPP+ to the medium there is a steep

voltage drop. The rate with which the TPP+ signal changes during calibration of the

electrode was always quicker than the fastest physiological response measured (uncoupling

of the mitochondria by addition of CCCP). Therefore the electrode response is both

kinetically competent and not rate limiting.

65

2.4.4 Respiratory measurements—The simultaneous measurement of oxygen uptake and

redox poise or oxygen uptake and membrane potential was achieved using an oxygen

electrode in combination with the Q-electrode or with the TPP+-electrode respectively. A

combined setup of all three electrodes will be discussed in chapter 3. All signal outputs

were recorded using a MacLab/200 (Macintosh Operating Systems, AD Instruments Pty

Ltd) and chart analysis software (Chart v3.6/s, AD Instruments Pty Ltd) on an iMac

desktop computer. Per assay 0.4-1 mg mitochondrial protein was added to 2.2 ml reaction

medium. Reaction media for yeast and plant mitochondria are osmotically different, see

section 2.1. Where the Q-electrode was employed, 1 µM ubiquinone-1 (Q1) was included in

the reaction medium. Where the TPP+-electrode was employed 1µM of TPP

+ was included

in the reaction medium. All experiments were done at room temperature.

In order to uncouple mitochondria the chemical carbonyl cyanide m-

chlorophenylhydrazone (CCCP) was added. With potato and arum a concentration of 1 M

was sufficient to completely uncouple the mitochondria; with yeast mitochondria however

it was found that in the presence of 1 M CCCP upon addition of substrate there was still

an upward deflection of the TPP+ signal which indicates the formation of . To

completely prevent the formation of the standard concentration of CCCP used with

yeast mitochondria was 2 M, twice the concentration used in previous work [93, 104].

2.4.4.1 Preparation of respiratory effectors—Unless otherwise stated respiratory substrates

and effectors were prepared in reaction medium (see section 2.1). For the yeast experiments

Ubiquinone-1, CCCP, antimycin A, octyl gallate and salicylhydroxamic acid were

dissolved in isopropanol which, unlike the more usual solvent ethanol, is not metabolised

by isolated S. pombe mitochondria and does not affect respiratory activity. For the plant

experiments ethanol was used.

66

2.4.4.2 Nomenclature—To indicate energetic conditions the conventions of Chance and

Williams [1] were used:

State 1: mitochondria alone in solution (S1)

State 2: mitochondria present + substrate (S2)

State 3: a limited amount of ADP added, leading to an increased vO2 (S3)

State 4: all ADP converted to ATP, vO2 decreases again (S4)

State 5: anoxia (S5)

Figure 2.9 shows a schematic representation of an oxygen trace with the different

bioenergetic states indicated. Mitochondria in vivo are believed to be in a state intermediate

between states 3 and 4 [162, 163].

Figure 2.9 Schematic representation of an oxygen uptake trace as recorded with an oxygen electrode.

67

2.4.4.3 Basic bioenergetic parameters—For characterisation of mitochondria the

respiratory control ratio (RCR) and the nmole of ADP molecules consumed per nmole of

oxygen atoms (ADP/O) were determined. A measure for the quality of the inner

mitochondrial membrane (IMM) in isolated mitochondria is the RCR. Usually the ratio of

the state 3 respiration rate (S3) divided by the state 4 respiration rate (S4) or the uncoupled

respiration rate divided by the state 2 respiration rate (S2) is used (steady state rates only).

The underlying idea being that under state 2 or state 4 conditions the IMM conductivity is

low. Upon addition of ADP or uncoupler the conductivity increases. If during isolation the

IMM is damaged, the state 2 or state 4 rate is relatively high due to the 'leakiness' of the

membrane which will result in a low RCR value. ADP/O values were determined according

to [164].

2.4.4.4 Q-pool kinetics—In this study a modular approach was used where components of

the ETC were lumped in two groups of modules: Q-pool reducing and Q-pool oxidising

pathways. The external NADH dehydrogenase or the combination of complex II with the

dicarboxylate carrier are the reducing pathways. The combination of complexes III and IV

combined with cytochrome c (cytochrome pathway) or the alternative pathway (which

consists of the alternative oxidase) are the oxidising pathways. Importantly, each unit

interacting with the Q-pool exhibits a kinetic dependence on the Q-redox poise. Since a

given steady state respiratory rate and corresponding level of Q-reduction represents the

situation where both ubiquinone reducing and ubiquinol oxidising pathway activities are

equal, it will reflect the respective kinetic dependencies of each of the Q(H2)-interacting

pathways involved.

By careful modulation of steady state respiratory activity, the kinetic dependence of

a particular pathway on the level of Q-reduction can be determined. This is done by

specifically altering the activity of one Q-interacting pathway at a time during respiratory

measurements so that the change in overall electron transfer (which is dependent upon the

activities of both Q-reducing and Q-oxidising pathways) will therefore depend on how the

opposing pathway changes with respect to the altered level of its substrate-product ratio

(the Q-redox poise). This was done by modulation of succinate dehydrogenase by titrating

with malonate (an inhibitor of SDH [165], titration range 0 – 5 mM) to obtain steady states

68

describing Q-oxidising pathway kinetics. Alternatively, respiratory activity was titrated

with small additions of subsaturating amounts of NADH (to a maximum of ~ 75 µM with

yeast mitochondria, to a maximum of ~100 M with potato mitochondria and to a

maximum of ~50 M with Arum mitochondria) which step-wise increased the activity of

the external NADH dehydrogenase [146] in the presence of ~ 10 units/ml of Glucose-6-

Phosphate dehydrogenase (EC 1.1.1.49) and 10 mM Glucose-6-Phosphate to ensure steady

state levels of NADH were maintained, as described in [146]. Additions of aliquots of

NADH had small or negligible effects on the Q-electrode (depending on the experimental

day). The Q-recordings were corrected for these effects according to [146].

Another method which was employed to obtain Q-oxidising pathway kinetics was through

the use of standard traces. In order to activate SDH in yeast mitochondria, both ATP and

glutamate are added. When done in sequence (as opposed to pre-incubation) this yields

three consecutive data points which are relatively spaced apart, i.e. they are not bundled

together in a small cluster. Finally, the modulation of the cytochrome pathway by inhibiting

respiratory activity using antimycin A (0 – 40 nM) was employed to obtain Q-reducing

pathway kinetics.

2.4.5 Modelling of Q-pool data—The fits used to describe the Q-pool kinetics presented

throughout this thesis were modelled according to van den Bergen et al. [76]. In this model,

each of the Q(H2)-interacting pathways is assumed to exhibit reversible Michaelis-Menten

kinetics in accordance with the following scheme:

where the forward reaction represents the oxidation of quinol substrate (S) to give quinone

product (P) catalysed by enzyme (E). This reaction scheme can be described by a simple

equation (for full derivation see appendix 1):

[2.6]

PE ES E S3

4

1

2

k

k

k

k

v s

s 1

69

When using this simplified rate equation in a v versus s plot, the value dictates the shape

of the kinetic curve. A positive value will give a convex curve indicating that the enzyme

has a higher affinity for substrate (QH2) than product (Q). Conversely, a negative value will

give a concave plot indicating that the enzyme has a higher affinity for product than

substrate. A straight line indicates that the affinity of the enzyme for substrate and product

is the same.

This equation has been used to model steady state rates of oxygen consumption and Q-

reduction levels that represent either Q-reducing or QH2-oxidising kinetics. In all of the

above expressions, the forward rate is defined as QH2-oxidation, therefore Q-reducing

enzymes will exhibit a negative value of v. For graphical convenience, all rates are plotted

in absolute terms, therefore, to model dehydrogenase kinetic data the equations sign is

reversed.

When fitting QH2-oxidising activity, the parameter is fixed at zero to reflect that there

will be no rate when s = 0 (the Q-pool is fully oxidised). In the case of Q-reducing activity

is fixed at – to reflect that there will be no rate when s = 1 (the Q-pool is fully reduced).

A respiratory steady state is reached when the rates through the reducing and oxidising

pathways are equal. The steady state Q-redox poise can readily be calculated by solving:

[2.7]

Where i indicates a summation over all involved enzymes. This model has been applied

with success in both plant [76] and yeast systems [70, 93].

Kinetic fits were made with Kaleidagraph version 3.02 which uses the least squared error

method (see chapter 9 in [166]).

01

i i

ii

s

70

2.6 Other methods

2.6.1 Spectroscopy—Reduction of ferricyanide (Fe(CN)63−

) was determined using a

spectrophotometer (CARY 400 Scan). Ferricyanide can accept electrons from cytochrome c

[1]. In the presence of KCN (1 mM), to inhibit complex IV, electron transfer chain activity

in isolated mitochondria can be measured spectroscopically by measuring the reduction of

ferricyanide (molar extinction coefficient = 1.02 x 103 M

-1 cm

-1 [167]). Absorption was

followed at 420 nm [168] (cuvette filled with reaction medium, mitochondria plus

substrates and inhibitors) from this value the signal of a ‘blank’ (cuvette filled with reaction

medium) was subtracted. Rates were expressed as nanomoles of ferricyanide reduced per

minute per milligram mitochondrial protein. The contents of the cuvette were kept

homogenous through the presence of a small stirring bean. Experiments were done at 25C.

2.7 Bioinformatic resources

The Wellcome Trust Sanger Institute http://www.genedb.org/

The Center for Biological Sequence Analysis http://www.cbs.dtu.dk/services/NetPhos/

(Technical University of Denmark DTU)

http://www.genedb.org/

http://www.cbs.dtu.dk/services/NetPhos/

71

Chapter 3

New insights into the regulation of plant succinate

dehydrogenase - revisited

3.1 INTRODUCTION

3.1.1 General background and aims—Complex II, otherwise known as succinate

dehydrogenase (SDH), is the only component of the electron transfer chain in mitochondria

which is also a component of the citric acid cycle and fulfils therefore a dual role, being

active in both the process of energy transduction and the generation of carbon intermediates

for biosynthetic metabolism [21] (see section 1.2.4 for a description of SDH). It is a well-

established fact that ATP can activate this protein [123, 124, 169-171], the mechanism of

which, at present, is still unclear. In previous work done in this laboratory it was

determined that ATP, ADP and oligomycin had a stimulatory effect on SDH in fresh potato

tuber mitochondria [21]. The stimulatory effect of the adenine nucleotides was suggested to

occur at the cytosolic side of the IMM. It was found that the effects of ATP and ADP were

not additive, suggesting that both nucleotides activate SDH in a similar fashion. It was

hypothesized that SDH was activated by these compounds indirectly, using the membrane

potential (∆) as an intermediate. On the basis of indirect evidence it was suggested that

inhibition of the plant mitochondrial K+

ATP channel (PmitoKATP) [172] upon addition of

adenine nucleotides could affect [21]. Activity of this channel, which is selective for

K+, can decrease the magnitude of . Both ADP and ATP have been shown to inhibit this

channel [172]. It was hypothesized that both ATP and ADP could potentially increase the

magnitude of through their inhibitory actions on PmitoKATP [21]. Addition of

oligomycin to potato mitochondria respiring on succinate is known to increase the

magnitude of [110]. A stimulatory effect of oligomycin on SDH could be reversed by

addition of uncoupler, which also implies a role for in the regulation of SDH [21].

Based on these observations it was suggested that SDH can be stimulated by an increase in

72

the magnitude of . This however was not demonstrated conclusively as was not

determined in that study (SDH regulation was investigated by simultaneous measurement

of oxygen consumption rate (vO2) and Q-pool redox poise (Qr/Qt) [21]). In this study a

TPP+- electrode [154] was used to determine ∆ under various energetic conditions. The

hypothesis that SDH in potato tuber mitochondria is activated by adenine nucleotides or

oligomycin indirectly via the membrane potential was investigated.

Fresh potato tuber mitochondria have been investigated extensively in the field of

bioenergetics and the respiratory kinetics of these mitochondria are well understood [76,

133, 139, 157, 162, 173]. Studies done with potato mitochondria focussed either on the

relationship between oxygen consumption rate and the membrane potential [111, 133, 139,

157, 173-178] or on the relationship between oxygen consumption rate and the Q-redox

poise [21, 76, 131]. One of the aims of this study was to simultaneously determine the

oxygen consumption rate, membrane potential and the Q-redox poise in isolated

mitochondria. In order to achieve this; an experimental set-up was used in which an

oxygen, a Q- and a TPP+-electrode were combined. The experimental approach in this

chapter is novel in the sense that three bioenergetic parameters were determined in isolated

mitochondria, either simultaneously by using a 3-electrode set-up or in parallel by doing

duplicate experiments with two set-ups: an oxygen and Q-electrode set-up and an oxygen

and TPP+-electrode set-up.

Affourtit et al. found that addition of adenine nucleotides or oligomycin to potato

mitochondria respiring on succinate led to increased oxygen consumption rates [21].

These results are at variance with findings obtained in an earlier study by Fricaud et al. in

the same laboratory [110, 111] where a decrease in oxygen consumption rates was

observed. Both findings were reproduced in the present study and an explanation will be

given in terms of the interplay between reducing and oxidising pathways showing that the

results obtained in these studies [21, 110, 111] are in agreement with each other. In light of

recent developments the involvement of the protonmotive force in the process of SDH

regulation will be reassessed. In order to appreciate the work presented in this chapter the

membrane potential in mitochondria and the regulation of SDH will be discussed in the

next two sections.

73

3.1.2 The membrane potential in mitochondria—In vitro, the magnitude of the membrane

potential in plant mitochondria is larger than in mammalian mitochondria. Typical state 4

values in plant mitochondria range between -200 to -240 mV [111, 139, 163, 175, 179-

182] although in some studies smaller values ~ -190 mV have been reported [172]. In

mammalian mitochondria the state 4 values are considerably smaller, in the range of –150

to -190 mV [132, 154, 163, 183-186]. State 4 membrane potential values in yeast

mitochondria are intermediate between those of mammals and plants with values in the

range of –180 to -200 mV [160, 187]. A common feature of all types of mitochondria is a

20 to 40 mV depolarisation which is seen upon addition of ADP (state 3) [132, 154, 160,

172, 175, 176, 181, 186].

It has been established that the protonmotive force in potato mitochondria mainly

consists of a membrane potential and that the pH component is negligible since addition

of nigericin, an ionophore catalysing the electroneutral exchange of H+ for K

+ [1] does not

lead to an appreciable change in [110, 139, 157]. It has also been reported that pH

values in plant mitochondria are very low [163]. The explanation for the lack of effect by

nigericin lies in the fact that plant mitochondria express a K+/H

+ exchanger [188] in the

IMM [157, 172]. Addition of nigericin to potato mitochondria isolated in this study did not

lead to any noticeable change in TPP+ signal and therefore p was assumed to be

equivalent to . Under ADP limited conditions it is found that mitochondrial respiration

can be substantially inhibited with only a small concomitant change in p [165, 189].

Figure 3.1 shows a typical vs. respiration rate relationship under ADP limited

conditions. The data in the plot are fictitious with respiration rate given in arbitrary units. In

a typical experiment, isolated mitochondria respiring on succinate are titrated with the

inhibitor malonate (cf. Figure 3 in [183]). It can be seen that the relationship between

electrical potential difference and current (respiration rate represents proton current across

the IMM [1]) is non-linear, which is usually referred to as being ‘non-ohmic’ [183]. A

small increase in at high values leads to a disproportional increase in respiration

rate. It is now commonly accepted that this behaviour is due to a non-ohmic increase in

proton permeability at high p [190-192] and that the non-ohmic relationship also holds for

other cations, such as K+, tetramethylammonium

+ and choline

+ [183]. Non-ohmic

current/voltage relationships have been determined in mammalian mitochondria [183, 189,

74

190, 193], plant mitochondria [139, 158], chromatophores [194] and bovine heart

submitochondrial particles [195].

Figure 3.1 Typical vs. respiration relationship in mitochondria under ADP limited conditions. A non-

ohmic relationship (see text) is seen. The data points are fictitious and respiration rate is given in arbitrary

units. Membrane potential values are given in absolute values as is done customarily in bioenergetics

literature. Figure adapted from Figure 3 in [183].

To initiate respiration in this study either succinate or NADH were added to the

medium containing the isolated potato mitochondria. Therefore is generated via

cytochrome pathway activity only (see section 1.2.1). Plant mitochondria express an

alternative oxidase (which is non-protonmotive, see section 1.2.11) and reducing

equivalents from succinate or NADH could bypass the cytochrome pathway which might

have an effect on . However, fresh potato tuber mitochondria do not display cyanide

resistant respiration [8, 76, 176] therefore the respiratory chain of the mitochondria used in

this chapter is identical to Figure 1.4 minus the alternative oxidase.

75

3.1.3 Regulation of SDH—SDH is a homotropic enzyme, succinate not only serves as a

substrate but also as a positive modifier of the protein. This activation was found in intact

mitochondria, membrane preparations and in the soluble, purified enzyme [10].

In this study SDH regulation was assessed in well coupled, intact mitochondria, in which

oxygen consumption rate, the Q redox poise and the membrane potential were determined.

The regulation of succinate dehydrogenase has been studied for over 50 years [20] and a

vast amount of literature is available (see [124, 196] and references therein). Interpreting

and comparing data from some of the older publications to the data obtained in this study is

not straightforward given the different types of mitochondrial preparations used several

decades ago when compared to contemporary standards. In the early literature which

focussed on mammalian SDH, Keilin-Hartree particles (using beef heart as source material)

were used [20, 197]. These particles were obtained by grinding heart tissue with sand

resulting in a mixture of damaged mitochondria and everted vesicles of inner mitochondrial

membrane [18], membrane fragments of other types of organelles are present in this type of

preparation as well [196].

SDH activity has been routinely determined spectrophotometically and via

determination of the oxygen consumption rate. A source of possible confusion is the

widespread use of malonate to activate SDH [20, 196, 198]. Malonate is known to be an

inhibitor of SDH [86, 133, 139] and has been used in studies to decrease the membrane

potential [133, 183] or the Q redox poise [76, 86, 104] in mitochondria respiring on

succinate. The binding of malonate to complex II, although it is a substrate competitor,

induces a conformational change from an inactive to an active form of the protein [10,

197]. Another inhibitor of SDH, oxaloacetate (OAA) [199] can tightly bound to SDH,

preventing the binding of substrate, leaving the protein inert [200]. Therefore when SDH is

inactive (SDH-OAA complex) it cannot be reduced by succinate [20]. Several agents can

aid in the displacement of oxaloacetate such as succinate, inosine di- and triphosphate and

malonate [200].

One widely used method to asses SDH activity is the phenazine methosulfate

(PMS) assay [201]. In this method SDH is reduced by succinate and oxidised by PMS.

Reoxidation of PMS is accomplished by 2,6-dichlorophenolindophenol (DCIP). Reduction

of DCIP is followed spectrophotometically at 600 nm. If SDH needs to be activated prior to

76

the PMS assay it can be incubated with malonate to dislodge OAA. Subsequently, an

aliquot of the solution is taken and transferred to the cuvette used in the PMS assay. To

prevent excessive inhibition upon carry over, a range between 1-4 mM malonate is

normally used [200]. A drawback of using the PMS assay in intact mitochondria is that the

IMM needs to be permeabilised which results in deenergized mitochondria [201]. Another

disadvantage is the use of artificial electron acceptors, which makes it difficult to compare

activity assays done spectrophotometically with assays done using an oxygen electrode.

It is well known that isolated SDH complexes have a lower turnover number

compared to SDH in its native environment. Upon reconstituting isolated SDH into the

IMM turnover numbers are restored [202, 203]. Extraction of quinones from inner

membranes results in a decreased turnover number of SDH whereas reinserting quinones

leads to restoration of the turnover number [204]. This agrees well with the observation that

ubiquinol is an activator of SDH [10]. Addition of externally added QH2 leads to activation

whereas addition of Q does not [124]. It has been reported that QH2 has a different binding

site than Q, binding of which leads to conformational change [205]. It was also found that

QH2 is not oxidised upon activation of SDH [124].

It is a well-established fact that SDH is activated by anions (ClO4- > I

- > Br

- > Cl

-)

[170, 206], this effect can be counteracted by OAA. It was determined that the anions

activate SDH directly, dislodging OAA, as opposed to an indirect chaotropic effect [124].

The ATP activation effect is well known [169] but its mechanism is still poorly

understood. Stimulation of SDH by ATP was shown to occur in the presence of cysteine

sulfinate (which transaminates OAA very effectively) in Baker’s yeast mitochondrial

particles. From this it was concluded that SDH activation by ATP does not occur through

removal of OAA [123]. ATP was found to activate SDH within 2-4 minutes, whereas full

activation by succinate was achieved only after 15-20 minutes [124]. The activation was

found to be not energy dependent as it was insensitive to oligomycin or uncoupler. ATP

activation is not seen in several types of enzyme preparations [124]. Whether or not ATP

interacts directly with SDH or if it exerts an effect on SDH indirectly via the membrane

potential will be investigated in this chapter.

77

3.2 RESULTS

3.2.1 General characterization—In this section the quality and general bioenergetic

characteristics of the isolated potato tuber mitochondria used in this study are assessed.

Figure 3.2 shows typical oxygen consumption (black) and (red) traces from an

experiment where both parameters were measured simultaneously. ATP (0.2 mM) was

incubated to fully activate SDH, the concentration needed to half maximally activate SDH

in potato mitochondria was determined to be 3 M [21]. Upon addition of succinate (5

mM) a full membrane potential (217 mV)10

is established (state 2)11

reflected by the

upward deflection of the TPP+ signal which indicates that TPP

+ molecules disappear from

the medium, drawn into the matrix, due to the build up of an electrical potential (the matrix

side of the IMM being negative with respect to the cytosolic side). Addition of ADP (0.1

mM) leads to a decrease in and a increase in oxygen consumption rate (vO2) which

demonstrates that respiration is coupled to phosphorylation (state 3). A downward

deflection of the TPP+ signal reflects expulsion of TPP

+ molecules from the matrix back

into the medium. When all ADP is depleted (state 4) and vO2 return to values

comparable to state 2. It can be seen that the amount of ADP added for the first state 3 is

not enough to generate a steady state , subsequently twice the amount of ADP is added,

which results in a proper state 3. After ADP is depleted again, and vO2 return to state 4

values. A final addition of ADP (0.2 mM) results in a state 3, which coincides with anoxia.

dissipates slowly, addition of an uncoupler, CCCP (1 M) leads to complete

dissipation and the TPP+ signal returns to the value before mitochondria were added. The

TPP+ axis does not show units for the membrane potential, as this parameter is not

measured directly, see section 2.4.3.

It has been reported that upon isolation, plant mitochondria are relatively deenergized and

do not generate a membrane potential in the absence of added substrate [139] as opposed to

rat liver mitochondria which display an initial low, rotenone sensitive, respiration rate

which runs out shortly after the start of an experiment [21]. This observation was confirmed

10

All membrane potential values are given in absolute values as is customarily done in the literature. 11

See section 2.4.4.2 for nomenclature used.

78

in our experiments (not shown) and it is concluded that potato mitochondria upon isolation

are depleted of endogenous substrates. With NADH as a substrate (oxidised by the external

NADH dehydrogenase, see section 1.2.3) traces similar to the ones in Figure 3.2 were

obtained, in contrast to SDH the external NADH dehydrogenase is fully active in the

absence of ATP. The membrane potential values obtained in this study agree well with

results previously obtained in this laboratory [38, 110, 111, 139] and with those of others

[157, 181]. Oxygen consumption rates with succinate as a substrate were found to be higher

than with NADH as a substrate which reflects differences in dehydrogenase kinetics [133].

Figure 3.2 Representative traces of oxygen consumption (black) and (red) measured in isolated potato

tuber mitochondria incubated with ATP (0.2 mM) to activate SDH. Additions: succinate (5 mM), ADP (0.1

mM), ADP (0.2 mM), ADP (0.2 mM) and CCCP (1 M). Amount of mitochondrial protein 0.7 mg.

Numbers in italics indicate steady state membrane potential values in mV, numbers in regular font indicate

steady state oxygen consumption rates in nmol O2 / min / mg protein.

Without ATP incubated a state 3 to state 4 transition is required to activate SDH. Figure 3.3

shows typical oxygen consumption (black) and Qr/Qt (red) traces from an experiment

where both parameters were measured simultaneously. Upon addition of succinate (5 mM)

SDH does not become fully activated which is reflected in the low Qr/Qt value (0.36)

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2

0

100

200

300

400

500

0 2 4 6 8 10 12

68

202

63

220

61

217

192

217

176

215

TP

P+ s

ign

al

(V) o

xy

ge

n (n

mo

l)

succinate ADP ADP ADP CCCPanoxia

time (min)

79

attained. This value agrees well with previous work done in this laboratory where a 10-35%

reduction of the Q-pool was found when succinate was added to potato mitochondria in the

absence of ATP [21, 207]. With ATP incubated, addition of succinate leads to a Q-

reduction level of 80-90% (data not shown). Addition of ADP (0.2 mM) leads to an

increase in vO2 (state 3) and a transient decrease followed by a slow increase in Qr/Qt as

SDH is seen to activate throughout state 3 due to the build-up of ATP that is being formed.

When all ADP is depleted, vO2 decreases (due to build up of p) and the Q-pool becomes

89% reduced, a value that agrees well with previously obtained results [21, 207]. Upon

addition of ADP (0.2 mM) a second state 3 is brought about which coincides with anoxia

leading to complete reduction of the Q-pool. NADH at high concentrations (2 mM) reacts

strongly with the Q-electrode therefore only representative traces with succinate are shown.

Figure 3.3 Representative traces of oxygen consumption (black) and Qr/Qt (red) measured in isolated potato

tuber mitochondria. Additions: succinate (5 mM), ADP (0.2 mM), ADP (0.2 mM). Amount of mitochondrial

protein 0.5 mg. Numbers in italics indicate steady state oxygen consumption rates in nmol O2 / min / mg

protein, numbers in regular font indicate Q redox poise expressed as the fraction QH2 in the pool.

0

100

200

300

400

500

600

0

0.2

0.4

0.6

0.8

1

0 2 4 6 8 10

ox

yg

en

(n

mo

l)

Qr/Q

t

time (min)

0.360.43

0.89

0.55

80

260

66

238

succinate ADPADP ADP anoxia

80

The RCR values, using the ratio of state 3 and state 4 oxygen consumption rates (see

2.4.4.3) for potato mitochondria respiring on succinate in this study were between 3 and 4

which agrees well with values obtained previously in this laboratory [21, 38, 207] and with

those obtained by others [157]. The RCR values indicate that the mitochondria are coupled

and that the isolation method yields intact mitochondria. The IMM is shown to function as

an isolating membrane, which is a requirement for an energy transducing system (see

postulate 4 on page 35). It can be seen in figures 3.2 and 3.3 that vO2 values decrease

throughout the trace upon successive state 3 to state 4 transitions, an explanation for this

will be offered later.

Mitochondria under ADP limited conditions show a non-ohmic vs. vO2

relationship, see section 3.1.2. Figure 3.4 shows the relationship between ∆ and vO2

under state 4 conditions in potato mitochondria respiring on succinate () or NADH ().

Succinate respiration was inhibited with malonate and NADH respiration was titrated

upwards using the NADH regenerating system (see section 2.4.4.4). It can be seen that both

sets of data overlap which indicates that there are no substrate dependent differences in

oxidizing pathway kinetics (see section 2.4.4.4 for a description of reducing and oxidising

pathway kinetics). The shape of the relationship between and vO2 is non-linear and

looks similar to the non-ohmic current/voltage relationships found previously in

mitochondria, cf. Figure 3.1.

Concluding, the isolation method used in this study yields well coupled potato

mitochondria of a quality comparable to that of previous studies done in this laboratory.

The respiratory kinetics agree well with what has been reported in the literature.

81

Figure 3.4 Combined state 4 cytochrome pathway kinetics of potato mitochondria respiring on either NADH

or succinate, vs. vO2. NADH () data were obtained using the NADH regenerating system. Succinate

() data were obtained with the malonate titration method. With succinate as a substrate a state 4 was

induced by adding an aliquot of ADP (50M) after the mitochondria were energised with succinate (5mM) +

ATP (0.2 mM), when ADP was depleted respiratory activity was titrated with malonate (up to ~ 4 mM).

Succinate titration data were obtained from one mitochondrial isolation, data points were taken from three

traces. NADH data were obtained from one mitochondrial isolation, data points were taken from two traces.

Respiration was initiated by addition of a sub-saturating amount of NADH (10 µM), an aliquot of ADP (50

µM) was added to induce a state 3 to state 4 transition, when ADP was depleted NADH was titrated using

sub-saturating amounts up to ~100 µM. Mitochondrial protein used per experiment was ~ 0.8 mg.

3.2.2 Stimulation of SDH by adenine nucleotides—Previous work done in this laboratory

demonstrated that SDH was activated by ATP in potato tuber mitochondria [21]. An

experiment was done to verify that the mitochondria used in this study displayed the same

behaviour. Figure 3.5 shows that upon addition of succinate the Q-pool redox poise (red

trace) increases up to 53% with a concomitant vO2 value of 77 nmol O2 / min / mg protein.

Addition of ATP (0.2 mM) leads to a further increase in Qr/Qt up to 85% with a

concomitant increase in vO2 to 107 nmol O2 / min / mg protein. Upon subsequent addition

of ADP (0.2 mM) a state 3 to state 4 transition is seen. These results are comparable to

those obtained previously in this laboratory [21, 207].

0

20

40

60

80

100

150 160 170 180 190 200 210 220 230

succinateNADH

vO

2 n

mo

l O

2 /

min

/ m

g p

rote

in

membrane potential mV

82

Having reproduced the original observation showing that ATP has a stimulatory effect on

SDH (reflected in both vO2 and Qr/Qt) in a next series of experiments the effect of adenine

nucleotides on the membrane potential in potato mitochondria respiring on succinate was

investigated.

Figure 3.5 ATP induced activation of SDH. Representative oxygen concentration (black) and Q-reduction

(red) traces illustrate the activation of SDH by ATP. At the points indicated by the arrows succinate (9 mM),

ATP (0.2 mM) and ADP (0.2 mM) were added. Mitochondrial protein used was 1 mg. Numbers in italics

indicate Qr/Qt values; numbers in standard font indicate vO2 values (nmol O2 / min / mg protein).

ATP was hypothesized to stimulate SDH indirectly, via a change in [21]. Upon

hydrolysis of ATP by complex V a membrane potential can be generated [1]. One

possibility is that the addition of ATP itself leads to the generation of which

subsequently activates SDH. This is hard to envisage under ADP limited conditions where

the high back pressure of p would prevent the hydrolysis of ATP [162]. However, as it

was found that incubation of ATP leads to full activation of SDH prior to addition of

succinate [207] it could be the case that generated by ATP hydrolysis activates SDH

0

100

200

300

400

500

600

0

0.2

0.4

0.6

0.8

1

0 0.8 1.6 2.4 3.2 4 4.8 5.6 6.4

ox

yg

en (

nm

ol)

Qr/Q

t

time (min)

succinate ATP ADP anoxia

0.53

0.84

0.71

0.9477

106

324

113

83

prior to the initiation of respiration by addition of succinate. It has been reported that

addition of ATP alone (no substrate present) to intact potato mitochondria cannot establish

a measurable membrane potential [162, 178]. This was confirmed in an experiment where

ATP was added to potato mitochondria in the reaction medium before addition of substrate,

see Figure 3.6. Upon addition of ATP (0.2 mM) a transient is generated with a peak

value of 166 mV. No concomitant changes were observed in vO2 or Qr/Qt, which indicates

that the transient was not generated due to ETC activity. Two minutes after dissipation

of the ATP induced , succinate (5 mM) is added, SDH is fully activated (as further

addition of ATP does not result in an extra increase in or vO2 , not shown). ATP does

not react with the TPP+

electrode and the transient seen in Figure 3.6 is not a measuring

artifact. With carboxyatractyloside (CAT), an inhibitor of the adenine nucleotide carrier

[126], or oligomycin incubated, no transient was seen upon addition of ATP which

indicates that the transient is indeed generated through ATP hydrolysis.

Figure 3.6 Addition of ATP to potato mitochondria in reaction medium leads to a transient .

Additions: ATP (0.2 mM) succinate (5 mM). Membrane potential values in mV. Mitochondrial protein used

was 0.6 mg.

It can be concluded from this experiment that SDH is fully activated by incubation with

ATP, as previously shown [207] and that the continuous presence of a is not a

1.2

1.3

1.4

1.5

1.6

1.7

0 1 2 3 4 5 6 7 8

TP

P+ s

ign

al V

time (min)

ATP succinate

166

225

84

requirement to keep the complex activated until the moment of succinate addition. One

interpretation of this would be that the transient activated SDH and that the protein

remained in an active configuration after was dissipated. This however is unlikely as it

was found by Affourtit et al. that deenergization of potato mitochondria respiring on

succinate (in the absence of ATP) led to deactivation of SDH [21].

It was hypothesized by Affourtit that addition of ATP leads to an increase in

stimulating SDH activity which is reflected by an increase in Qr/Qt and vO2, see Figure 3.5

and compare figures 1 and 2 from [21]. The experiment from Figure 3.5 was repeated, this

time determining oxygen consumption rate and the membrane potential simultaneously, see

Figure 3.7. Upon addition of succinate a 224 mV membrane potential is generated with a

concomitant vO2 rate of 77 nmol O2 / min / mg protein. Addition of ATP (0.2 mM) leads to

an increase in ∆ up to 230 mV with a concomitant increase in vO2 to 83 nmol O2 / min /

mg protein. Upon subsequent addition of ADP (0.2 mM) a state 3 to state 4 transition is

seen. This experiment shows the assumption made by Affourtit et al. [21] to be correct.

Addition of ATP to potato mitochondria respiring on succinate leads to an increase in .

When ATP was pre-incubated a maximum (~220-230 mV) upon addition of succinate

was attained, further additions of ATP had no effect on the membrane potential (data not

shown). It was also found by Affourtit et al. that ADP had a stimulatory effect on SDH (cf.

Figure 2A in [21]). Addition of ADP to mitochondria in the absence of CAT leads to a

decrease in due to phosphorylation (cf. figures 3.2 and 3.7). To avoid this, potato

mitochondria were incubated with CAT12

and under these conditions the addition of ADP

to mitochondria respiring on succinate was found to stimulate SDH to an even higher extent

than ATP [21] (Qr/Qt and vO2 measured simultaneously). Through inhibition of the

adenylate kinase ((by incubating with Ap5A) ADP was prevented from being converted

into ATP and it was concluded that the stimulatory effect was due to ADP per se. The

experiment was repeated, this time measuring the membrane potential and oxygen

consumption rate simultaneously, see Figure 3.8.

12

Addition of CAT to potato mitochondria respiring on succinate does not lead to a change in (data not

shown).

85

1.6

1.7

1.8

1.9

2

2.1

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23077

83

anoxia

Figure 3.7 ATP induced activation of SDH. Representative oxygen concentration (black) and membrane

potential (red) traces illustrate the activation of SDH by ATP. At the points indicated by the arrows succinate

(9 mM), ATP (0.2 mM) and ADP (0.2 mM) were added. Membrane potential values (italic) in mV, oxygen

consumption rate values (regular font) in nmol O2 / min / mg protein. Mitochondrial protein used was 0.8 mg.

Figure 3.8 ADP induced activation of SDH in the presence of CAT (10 M). Representative membrane

potential trace illustrating the activation of SDH by ADP. At the points indicated by the arrows succinate (9

mM) and ADP (0.2 mM) were added. Membrane potential values in mV. Mitochondrial protein used was 0.6

mg.

1.1

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ADPsuccinate

86

With CAT (10 M) incubated, upon addition of succinate (5 mM) a membrane potential of

~204 mV was established, addition of ADP (0.2 mM) led to a further increase up to 220

mV. This result again confirmed Affourtit’s assumption [21] that addition of adenine

nucleotides (CAT incubated when ADP is used) leads to an increase in .

3.2.3 Are the effects of ATP on , Qr/Qt and vO2 simultaneous?—The experiments

shown thus far were done with either a combination of an oxygen with a Q-electrode or

with a combination of an oxygen with a TPP+-electrode. One of the aims of this thesis work

was to set up a combined system of an oxygen, a Q and a TPP+-electrode in order to

determine three parameters simultaneously, vO2, Qr/Qt and . The reaction vessel used

allows for multiple electrodes to be fitted, see Figure 2.1. The Q-electrode (platinum and

glassy carbon electrodes) and the TPP+-electrode were fitted in the reaction vessel. To the

same vessel two reference electrodes (see Figure 2.7A) were fitted. The presence of the

TPP+-electrode did not lead to any interference with the Q-electrode. However the TPP

+-

electrode baseline signal shifted by about 3 V (increase) when the Q-electrode was

switched on. Apart from displacing the baseline signal by a fixed value no interference with

the TPP+-electrode was seen. In control experiments using isolated fresh potato tuber

mitochondria respiring on succinate it was determined that all electrodes operated

independently and no electrical cross-talk was observed, i.e. recordings made with

individual electrodes looked the same as recordings made with combinations of electrodes.

With a working system it was now possible to simultaneously measure three bioenergetic

parameters in isolated mitochondria. Figure 3.9 shows that upon addition of ATP to potato

mitochondria respiring on succinate the changes in vO2, Qr/Qt and occur

simultaneously13

.

Setting up the 3-electrode system was successful and recordings were made with

both potato and S. pombe mitochondria. It was however decided not to use it as the standard

technique. Setting up the system is a delicate matter. Both the Q-electrode and the TPP+-

electrode signals need some time to stabilise at the beginning of the experiment. Then the

TPP+-electrode needs to be calibrated which is a time consuming process.

13

Given that the change in oxygen consumption rate is small it cannot be appreciated from the oxygen

concentration curve without looking at the first derivative of this trace.

87

Figure 3.9 Activation of SDH by ATP. Three parameters: oxygen concentration (black), Qr/Qt (red) and

(blue) measured simultaneously. Additions: succinate (5 mM) ATP (0.2 mM). Mitochondrial protein used

was 0.7 mg.

The Q-electrode can be volatile in its behaviour and has at times the tendency to show drift

at the end of a trace when anoxia is reached. Because of this no values can be assigned to

the Q recording as the Q-signal under anoxia is used as a reference value.

Problems with the Q-electrode arose more often in the 3-electrode setup than with the

combination of just the oxygen and Q-electrode. It was deemed more effective to do

duplicate experiments with two set-ups (oxygen-Q and oxygen-TPP+) on the same day with

the same mitochondrial preparation and combine the results. Determining vO2, Qr/Qt and

in parallel was the standard way of doing experiments throughout the remainder of this

work. The results shown in figures 3.7-3.9 indicate that addition of adenine nucleotides to

potato mitochondria respiring on succinate does indeed lead to an increase in . But this

in itself does not prove that it is the change in which stimulates SDH.

Figure 3.10 shows a modular representation (see section 1.5) of the ETC of fresh

potato mitochondria respiring on succinate. There is only one Q-reducing pathway (SDH in

combination with the dicarboxylate carrier) and one Q-oxidising pathway (the cytochrome

pathway). Arrows indicate the rates of Q-pool reduction (a) and Q-pool oxidation (b). In

88

terms of this model three hypotheses can be formulated to explain the observations so far

described:

1) Addition of ATP or ADP (in the presence of CAT) leads to an increase in through

interaction with a component which is outside the defined system (essentially Affourtit’s

hypothesis, inhibition of PmitoKATP [21], see section 3.1.1). The increase in stimulates

SDH, this leads to an increase in rate a and a concomitant increase in reduction of the Q-

pool, as an effect of this, rate b will also increase which leads to an increased vO2 (cf.

Figure 3.5).

2) Addition of ATP or ADP (in the presence of CAT) leads to a direct stimulation of SDH,

this leads to an increase in rate a and a concomitant increase in reduction of the Q-pool, as

an effect of this rate b increases which leads to increased activity of the cytochrome

pathway. The electron transfer rate through complexes III and IV increases and so does the

rate of proton translocation. is a passive follower of events and not an effector in this

case.

3) Addition of ATP or ADP (in the presence of CAT) has a dual effect. Both SDH and

are affected, which leads to a simultaneous change in both reducing and oxidising pathway

kinetics.

Figure 3.10 Modular representation of the ETC in potato mitochondria respiring on succinate, see text.

To test hypothesis 1 experiments were done in the presence of an uncoupler.

89

3.2.4 Stimulation of SDH by adenine nucleotides in the presence of uncoupler—In the

following experiment the effect of ATP on SDH activity was assessed in the presence of

uncoupler, see Figure 3.11. In the absence of ATP addition of succinate (5 mM) generates a

membrane potential of 210 mV. Addition of an uncoupler increases proton permeability of

the IMM which leads to dissipation of p. Because of this the activity of the ETC increases

in a futile attempt to restore p which is concomitant with rapid respiration [1]. It can be

seen that upon addition of CCCP (1 M) is dissipated but vO2 only increases 1.4 times

in contrast to what is seen when ADP is added (cf. Figure 3.3) where vO2 is increased ~3.3

times. This suggests that SDH is partially deactivated. Subsequent addition of ATP (0.2

mM) approximately 2 minutes later results in an increased respiration rate (~2.8), but no

change in is seen. The same experiment but with ADP (0.2 mM) instead of ATP gives

similar results (data not shown).

Figure 3.11 Stimulatory effect of ATP on SDH in the presence of uncoupler. Representative (red) and

oxygen consumption (black) traces. Additions: succinate (5 mM), CCCP (1 M) and ATP (0.2 mM).

Membrane potential values in mV, oxygen consumption rate values in nmol O2 / min / mg protein.

Mitochondrial protein used was 0.5 mg.

1.4

1.45

1.5

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1.65

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210

82

115

324

succinate CCCP ATP

90

Affourtit hypothesized that the activity of SDH by adenine nucleotides is modulated via the

protonmotive force [21] but could not demonstrate this, concluding that: “the data

presented in this paper remain inconclusive as to whether activation of SDH by adenine

nucleotides is also wholly mediated by the protonmotive force” (taken from [21]).

This question can now be answered conclusively, Figure 3.11 demonstrates that addition of

ATP to uncoupled potato mitochondria respiring on succinate leads to activation of SDH

but not to a change in . Based on these results it was concluded that adenine nucleotides

under uncoupled conditions can stimulate SDH directly. Hypothesis 1 can be discarded.

Hypothesis 2 can explain the results shown thus far, a stimulatory effect of adenine

nucleotides on SDH leads to both an increase in Qr/Qt and , with the latter being a

passive follower of events. Further investigation of the modulatory role of on SDH

stimulation will reveal that hypothesis 2 is also incorrect.

3.2.5 Stimulation of SDH by oligomycin—Affourtit et al. found an activating effect on SDH

by oligomycin (an inhibitor of the ATP synthase, see section 1.2.9). Addition of oligomycin

to fresh potato mitochondria respiring on succinate (no ATP incubated) results in a large

increase in Qr/Qt (cf. Figure 3A in [21]) which is concomitant with a small but significant

increase in vO2 [21]. This was interpreted as a stimulation of SDH, however, upon addition

of uncoupler, (which dissipates p) the respiratory rate did not increase. This suggests that

the oligomycin induced activation of SDH was reversed by uncoupler, or that SDH was not

activated in the first place at all. A similar effect was seen with N,N’-

dicyclohexylcarbodiimide (DCCD) another inhibitor of complex V [21], suggesting that

oligomycin has no direct effect on SDH. The activating effect of oligomycin on SDH was

not to the same extent as that by ATP, maximal activation by oligomycin was ~ 85% of the

stimulation observed at saturating ATP concentrations [21]. Previous work done in this

laboratory by Fricaud et al. has shown that addition of oligomycin (and other ATP synthase

inhibitors such as efrapeptin and aurovertin) to potato mitochondria respiring on succinate,

in the absence of ATP, results in an increase in magnitude of (~ 20 mV) [110].

91

Figure 3.12 Effect of oligomycin on potato mitochondria respiring on succinate. Representative (blue)

Qr/Qt (red) and oxygen concentration (black) traces, measured simultaneously using the 3-electrode system.

Additions: succinate (5 mM), oligomycin (1 M). Amount of protein used was 0.7 mg.

Figure 3.12 shows a representative experiment where oxygen consumption (black), Qr/Qt

(red) and (blue) were determined simultaneously (using the 3-electrode setup).

Upon addition of succinate (5 mM) a of 214 mV was established, with a concomitant

vO2 of 66 nmol O2 / min / mg protein and a Qr/Qt of 24%. Addition of oligomycin (1 M)

led to an increase in magnitude of to 230 mV (agreeing well with results by Fricaud et

al. [110]) with a concomitant increase in Qr/Qt to 80 % (agreeing well with results by

Affourtit et al. [21]) vO2 however decreased slightly, to 60 nmol O2 / min / mg protein. The

results in Figure 3.12 confirm both Affourtit’s and Fricaud’s earlier findings showing that

addition of oligomycin leads to an increase in Qr/Qt and an increase in respectively, in

potato mitochondria respiring on succinate.

The observed decrease in vO2 is at variance with Affourtit’s findings [21] but in

agreement with results from Fricaud et al. [110, 111] who found that upon addition of

oligomycin to potato mitochondria respiring on succinate, respiration was inhibited by 31%

[111]. Interestingly enough it was found in the same study that addition of ATP (0.25 mM)

to potato mitochondria respiring on succinate also led to inhibition of respiration (by 25%)

[111].

92

Inhibitory effects of ATP and oligomycin on respiration were also seen with NADH as a

substrate [110] which indicates that these effects are not specific for succinate dependent

respiration. This suggests that the inhibitory actions of ATP and oligomycin exert their

effects at the cytochrome pathway side. The only apparent difference between the

experiments of Affourtit and Fricaud is the time interval between the addition of succinate

and the addition of either ATP or oligomycin. In Affourtit’s experiments ATP or

oligomycin were added typically within 1-3 minutes after respiration was initiated by the

addition of succinate, whereas in Fricaud’s experiments this interval was between 5-7

minutes. Potato mitochondria in this study were isolated using the exact same protocol as

used by Affourtit14

(see section 2.1.3) and RCR values are in the same range (~3-4) as in

Affourtit’s study [153]. The results shown so far are in good agreement with results

obtained by Affourtit previously [21] and it is therefore assumed that the quality and the

respiratory kinetics of the potato mitochondria in this study are essentially the same as

those in [21]. The RCR values from the study of Fricaud are in the range of 3-4 as well.

The protocol for isolating potato mitochondria used by Fricaud [38] is somewhat different

to the one used by Affourtit and this author. There are some small differences in

concentration of some of the chemicals in the grinding medium:

Chemical Affourtit / this study Fricaud

MOPS 40 mM 20 mM

cysteine 10 mM 7 mM

EDTA 2 mM 10 mM

and in the reaction medium:

Chemical Affourtit / this study Fricaud

MOPS 20 mM 10 mM

MgCl2 1 mM 5 mM

KH2PO4 5 mM 10 mM

14

Dr Affourtit personally supervised the isolation of the potato mitochondria during the initial stage of this

D. Phil project

93

More importantly though, in Fricaud’s study [38] succinate (1 mM) was added to the

washing medium to minimise SDH inactivation. Possibly the differences in respiratory

kinetics between the studies of Affourtit and Fricaud were due to slight differences in the

method of isolation. Since the potato mitochondria used in this study are essentially the

same as those used by Affourtit (and are therefore expected to behave in a similar way) this

hypothesis was tested by repeating some of the experiments done by Fricaud et al.

3.2.6 Adenine nucleotides and oligomycin inhibit succinate dependent respiration in potato

mitochondria—It was shown that addition of ATP or oligomycin to potato mitochondria

respiring on succinate leads to an inhibition of respiration [110]. It was already shown that

addition of oligomycin leads to a decrease in vO2, see Figure 3.12. To investigate the

effects of ATP on succinate dependent respiration, experiments were done using the

oxygen electrode. In order to appreciate changes in oxygen consumption rate it is useful to

take the first derivative of the oxygen concentration trace. Figure 3.13 shows the slow

activation of SDH by succinate (SDH is a homotropic enzyme), the oxygen concentration

(black) decreases slowly until anoxia is reached and the oxygen consumption rate (red) is

seen to increase slowly over time until after ~9 minutes a steady state rate is reached. Since

oxygen concentration decreases over time the derivative of this trace has only negative

values.

94

Figure 3.13 SDH activation by succinate in potato mitochondria. Representative oxygen consumption trace

(black) and its first derivative (red) which represents the oxygen consumption rate. Succinate was added to

initiate respiration (5 mM), mitochondrial protein used is 0.8 mg.

To test the effect of ATP on vO2, experiments were done where ATP was added to potato

mitochondria respiring on succinate either 2 or 6 minutes after respiration was started with

succinate. Figure 3.14 shows the time dependent effects of ATP on succinate dependent

respiration in potato mitochondria. Succinate (5 mM) was added to initiate respiration, ATP

(0.2 mM) was added after 2 minutes (A) or 6 minutes (B). It can be seen in Figure 3.14A

that upon addition of ATP vO2 stabilises (at ~50 nmol O2 / min / mg protein) whereas in

3.14B it is clearly inhibited (stabilising at ~45 nmol O2 / min / mg protein). Interestingly

enough the final vO2 reached with succinate alone (~87 nmol O2 / min / mg protein) is

higher than with ATP present (data not shown).

0

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in / m

g p

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in

time (min)

succinate anoxia

95

Figure 3.14 Time dependent effects of ATP on succinate dependent respiration. Oxygen consumption (black)

and its first derivative (red). Succinate (5 mM) was added to the medium to initiate respiration, ATP (0.2 mM)

was added 2 minutes (A) or 6 minutes (B) after succinate addition, traces done with same mitochondrial

preparation, mitochondrial protein used is 0.8 mg.

250

300

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500

550

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B

96

Steady state vO2 values from a series of duplicate experiments are given in Table 3.1.

Mitochondria were well coupled (RCR 4.6)15

and the respiratory rate under ADP limited

conditions was relatively low.

Table 3.1 Averaged steady state oxygen consumption rates of potato mitochondria respiring on succinate.

Rates obtained from one mitochondrial isolation, averaged values from duplicate experiments.

Concentrations: succinate (5 mM), ATP (0.2 mM). Mitochondrial protein used was 0.8 mg.

From Table 3.1 it can be seen that addition of ATP has an inhibitory effect on succinate

dependent respiration in potato mitochondria as the highest oxygen consumption rate is

reached when only succinate is present. As described in section 3.1.3, SDH is a homotropic

enzyme, i.e. succinate is not only substrate but also activator. Activation of SDH by

succinate is a slow process and can take up to 15-20 minutes [124]. It can be seen in Figure

3.13 that vO2 starts accelerating the moment succinate is added and after approximately 9

minutes a steady state rate is reached. In Figure 3.14A it can be seen that upon addition of

ATP acceleration stops immediately and a steady state oxygen consumption rate is reached.

Taking the average oxygen consumption rate prior to and after addition of ATP would lead

one to conclude that addition of oxygen consumption is increased upon addition of ATP.

When comparing Figure 3.14A with Figure 3.13 it becomes apparent that addition of ATP

in fact limits succinate dependent respiration. This does not disprove however that ATP

stimulates SDH activity in potato mitochondria, Figure 3.11 clearly shows that in the

presence of uncoupler ATP activates SDH. It was also shown by comparison of reducing

pathway kinetics [21] that SDH activity is higher in the presence than in the absence of

ATP.

15

state 3 rate / state 4 rate

Additions averaged steady state vO2

(nmol O2 /min /mg protein)

succinate 87

succinate + ATP (added after 2 min) 50

succinate + ATP (added after 6 min) 45

97

It was shown by Fricaud et al. that addition of ATP led to inhibition of succinate dependent

respiration [110]. The effects of ATP addition on however were not determined. Figure

3.7 showed that addition of ATP, within 3 minutes after addition of succinate, leads to an

increase in . The averaged vO2 after addition of ATP is also increased slightly. An

experiment was done where ATP (0.2 mM) was added ~6 minutes after initiation of

respiration with succinate (5 mM), see Figure 3.15. It can be seen clearly that increases

upon addition of ATP, vO2 however decreases considerably. The same experiment was

repeated, this time measuring vO2 and Qr/Qt simultaneously, see Figure 3.16. Again it can

be seen that upon addition of ATP (~6 minutes after initiation of respiration with succinate)

Qr/Qt increases (cf. Figure 3.5) vO2 however decreases considerably.

It was reported by Fricaud et al. that ATP and oligomycin also had an inhibitory

effect on NADH dependent respiration [110], suggesting that these effects are substrate

independent. Experiments with the oxygen electrode revealed that in potato mitochondria

respiring on NADH addition of either ATP or oligomycin led to a decrease in oxygen

consumption rates (data not shown), comparable to [110].

Figure 3.15 Inhibition of succinate dependent respiration by ATP. Representative vO2 (black) and (red)

traces. Respiration was initiated with succinate (5 mM), ATP (0.2 mM) was added ~6 minutes later.

Amount of mitochondrial protein used was 0.8 mg.

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98

Figure 3.16 Inhibition of succinate dependent respiration by ATP. Representative vO2 (black) and Qr/Qt

(red) traces. Respiration was initiated with succinate (5 mM), ATP (0.2 mM) was added ~6 minutes later.

Amount of mitochondrial protein used was 0.5 mg.

The results from figures 3.13-3.16 indicate that the differences in respiratory kinetics

between the studies of Affourtit and Fricaud are not due to a difference in isolation of the

mitochondria since Fricaud’s findings could be reproduced in potato mitochondria which

are essentially the same as the mitochondria used in Affourtit’s study. Figures 3.14 to 3.16

indicate that with mitochondria from the same preparation one can get different responses

upon addition of ATP.

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99

3.3 DISCUSSION

In this study a modulatory role of the protonmotive force on SDH regulation in fresh potato

tuber mitochondria was investigated. In a previous study done in this laboratory the

activation of SDH by adenine nucleotides was quantified in terms of Q-redox poise and

oxygen consumption rates [21]. It was believed that oligomycin also had an activating

effect, which was unexpected. It was hypothesized that SDH was activated indirectly by

adenine nucleotides using p as an intermediate. Evidence was given suggesting a possible

role for the mitochondrial K+

ATP channel in this process [21].

The potato mitochondria isolated in this study are of good quality and the

respiratory kinetics agree well with what has been found previously in this laboratory [21,

110] and by others [157], (figures 3.2-3.4). The stimulatory effect of adenine nucleotides on

SDH (in terms of Qr/Qt and vO2) as found by Affourtit et al. [21] was reproduced (Figure

3.5). It was previously shown in this laboratory that incubation in the presence of ATP fully

activates SDH in potato mitochondria [207]. It was shown that addition of ATP to potato

mitochondria in the absence of substrate leads to a transient which was generated by

the ATP synthase via hydrolysis of ATP (Figure 3.6). The continuous presence of was

not a requirement to keep complex II activated until the moment of succinate addition.

Possibly the transient activated SDH which remained in an active configuration after

was dissipated. This is unlikely however as it was determined that deenergization of

potato mitochondria respiring on succinate (in the absence of ATP) led to a deactivation of

SDH [21]. It was hypothesized originally by Affourtit et al. [21] that addition of ATP

would lead to an increase in . This was confirmed in an experiment where was

determined using a TPP+-electrode [154] (Figure 3.7).

It was determined that in the presence of CAT, addition of ADP to potato mitochondria

respiring on succinate leads to stimulation of SDH in a way similar to stimulation by ATP

[21]. Through inhibition of the adenylate kinase ADP was prevented from being converted

into ATP and it was concluded that the stimulatory effect was due to ADP per se.

Subsequent addition of ATP does not lead to any further significant stimulation of SDH (cf.

Figure 2A in [21]), this suggests that both adenine nucleotides activate SDH via the same

mechanism [21]. Addition of ADP to potato mitochondria respiring on succinate, incubated

100

in the presence of CAT was found to increase (Figure 3.8), again confirming the

hypothesis by Affourtit that addition of adenine nucleotides16

can lead to an increase in .

Using a 3-electrode set-up it was possible to determine vO2, Qr/Qt and

simultaneously. It was shown in potato mitochondria respiring on succinate that upon

addition of ATP the parameters , Qr/Qt and vO2 all change simultaneously (Figure 3.9).

The 3-electrode system performed adequately but proved in practice too volatile to use

effectively for experimental work. It was decided to do duplicate experiments using two

set-ups (oxygen-Q and oxygen-TPP+) using mitochondria that were isolated the same day.

It was shown that in the presence of uncoupler (p dissipated) the addition of ATP

or ADP could stimulate SDH (Figure 3.11). Therefore the presence of is not required

for adenine nucleotides to activate SDH. This does not exclude the possibility that under

coupled conditions there could be an indirect effect of adenine nucleotides on SDH

activation via a change in . It was found that a decrease in by addition of uncoupler

leads to deactivation of SDH (cf. Figure 3A in [21]) which suggests that an increase in

might activate SDH. Affourtit discusses several mechanisms via which the addition of

adenine nucleotides could lead to an increase in magnitude of in potato mitochondria

[21]:

A possible role for the uncoupling protein:

ATP could have an inhibitory effect on the mitochondrial plant uncoupling protein (UCP,

see section 1.2.10). Potato tuber mitochondria express an uncoupling protein [44, 180,

208]. Its activity leads to an increased proton conductivity of the IMM which could lead to

a decrease in the magnitude of . It is known that mammalian UCP activity can be

inhibited by purine nucleotides [1]. Borecký et al. expressed the Arabidopsis thaliana

uncoupling protein in E. coli cells and found that both ATP and ADP could inhibit its

activity [209]. The effects of ADP and ATP on the membrane potential in potato

mitochondria could be explained in terms of UCP inhibition. After Affourtit’s study was

published however it was found that in potato mitochondria, addition of ATP had no effect

16

mitochondria incubated in the presence of CAT when ADP is added.

101

on fatty acid induced uncoupling [179], i.e. UCP activity in potato mitochondria is

insensitive to the addition of ATP (it is inhibited by GTP [210]). Therefore the increase in

caused by ATP addition to potato mitochondria could not have occurred through the

inhibition of UCP.

A possible role for CAT-insensitive transport of adenine nucleotides:

SDH in potato mitochondria respiring on succinate, incubated in the presence of CAT, is

activated by either ATP or ADP, this suggests that the activation occurs at the cytosolic

side of the IMM. This interpretation was not fully conclusive though as ATP might enter

the matrix via a CAT-insensitive nucleotide carrier [21]. The mitochondrial ATP-Mg/Pi

transporter was reported to catalyse the exchange of ATP-Mg for Pi [211].

The name ATP-Mg/Pi transporter was later found to be a misnomer as this protein can in

fact exchange AMP and ADP as well [212]. The exchange of adenine nucleotides for

phosphate is electroneutral and occurs in the presence of CAT [212]. An ortholog of this

protein has been putatively identified in Arabidopsis thaliana [212]. Experiments done by

Leach in a previous study in this laboratory [207] suggest however that CAT-insensitive

nucleotide transport does not occur in potato mitochondria. In the absence of both Mg2+

and

Pi and presence of CAT addition of ATP led to activation of SDH. This suggests strongly

that ATP activates SDH on the cytosolic side and does not cross the IMM in a CAT-

insensitive way. If ADP were to be transported by the ATP-Mg/Pi transporter in the

presence of CAT this could activate the ATP-synthase (inducing a state 3) which would

result in a decrease of Qr/Qt and , but this is not observed, see Figure 3.8 and compare

figures 1B and 2A in [21].

A possible role for the K+-ATP channel:

The existence of a mitochondrial K+

ATP channel in plants (PmitoKATP) has been

demonstrated [172]. This channel allows K+ ions to flow into the matrix thereby

depolarising the existing across the IMM. This channel can be inhibited by both ATP

and ADP [172] which leads to an increase in . SDH is stimulated by ATP and ADP and

102

is possibly deactivated by a decrease in . Given the parallels between both processes,

suggesting a relationship between PmitoKATP inhibition and SDH activation is very

reasonable. This channel was found to be selective for K+ and Cs

+ which both could

collapse in respiring mitochondria, whereas Na+ and Li

+ had no effect [172], therefore

in order for this channel to be able to exert an effect on , potassium has to be present in

the medium. It has been reported however that the flux through PmitoKATP is too small to

decrease significantly (only 1 to 2 mV) [213]. Affourtit claims that “when

mitochondria are incubated in the presence of KCl, succinate alone is incapable of

generating a ” referencing [172]. This however is not true. Work done previously in this

laboratory and by others, demonstrates that potato mitochondria, incubated in the presence

of KCl, generate a upon addition of succinate [38, 110, 111, 139, 175, 181, 210]. In

this study it was also found that potato mitochondria incubated in the presence of KCl

generate a membrane potential upon addition of succinate, see figures 3.7-3.9, 3.11, 3.12

and 3.15. Upon reading [172] it also becomes apparent that Pastore et al. never claim that

in mitochondria incubated in the presence of KCl succinate is incapable of generating a

. Figure 2D in [172] clearly shows that durum wheat mitochondria incubated in the

presence of various concentrations of KCl generate a upon addition of succinate. Only

at a high concentration of KCl (60 mM) is the generation of a prevented. What the

article does show is that in the presence of ATP, potassium induced depolarisation can be

partially prevented (cf. Figure 2B in [172]).

To investigate a possible role of PmitoKATP in the activation of SDH by adenine

nucleotides the role of K+

in SDH activation was studied [21]. Figure 3.16 shows a figure

taken from [21] (Figure 4A) in which an effect of K+ on SDH activation can be clearly

seen. Reduction of the Q-pool in time was measured in potato mitochondria under various

energetic conditions. At T = 0 succinate was added to the reaction medium. Experiments 1,

3 and 5 were performed in potato reaction medium (see section 2.1.3.2), which contains K+;

experiments 2, 4 and 6 were performed in potato reaction medium, but with Na+ substituted

for K+. Trace 1 shows time resolved Q-reduction upon addition of succinate; no steady state

is reached before anaerobiosis. In the absence of K+ a steady state is reached quickly, trace

2. When incubated with the uncoupler CCCP, SDH is not activated at all irrespective of the

medium used, traces 3 and 4. When incubated with ATP SDH activation is maximal, traces

103

5 and 6. A clear effect of K+ on SDH activation is demonstrated by the difference in traces

1 and 2. However, this effect is not related to an effect of the PmitoKATP as indicated by

experiments 5 and 6. ATP is assumed to activate SDH indirectly via which is assumed

to increase due to inhibition of the PmitoKATP by ATP. In the absence of K+ in the medium

PmitoKATP will be inactive and has no influence on . Inhibiting PmitoKATP in the

absence of K+ therefore will have no effect on and trace 5 should look similar to trace

1. SDH activation cannot not occur via inhibition of PmitoKATP.

Figure 3.16 The effect of K+ on SDH activation. Figure 4A from [21], see text for details.

Concluding, also under coupled conditions PmitoKATP is not involved in the process of

SDH activation. K+

has been found to directly inhibit SDH [214], it was suggested that

potassium induces a conformational change of the enzyme to an inactive form. Other salts

such as NaCl, choline chloride and NaNo3 were shown to have a similar inhibitory effect. It

was also found previously that anions activate SDH [170, 206, 215]. Perhaps these ion

induced changes in SDH activation are due to direct electrostatic effects and Affourtit’s

potassium experiments show a direct inhibitory effect of K+

on SDH. It has been found that

anions favour dislodgement of OAA from SDH [124]. Possibly the presence of potassium

favours binding of OAA to SDH.

104

SDH activity has been shown to be dependent on the protonmotive force [21, 169, 216].

The experimental results presented in this chapter suggest that ATP and ADP can activate

SDH in the absence of which indicates that SDH activity is not modulated by

exclusively which leaves the possibility open that under coupled conditions Affourtit’s

hypothesis could still be true. After investigating the mechanisms with which adenine

nucleotides could affect in potato mitochondria, taking into account recent work that

was published after Affourtit’s publication the possibility that ADP and ATP can stimulate

SDH indirectly via seems unlikely.

ADP and ATP most likely interact directly with SDH. It was shown that both

nucleotides stimulate SDH cytosolically [21]. Based on its known structure (see figures 1.5

and 1.6) the only tentative binding sites for adenine nucleotides could be located on the

small fragments of subunits C and D which extend into the IMS. A phosphorylation site on

the SDH flavoprotein in potato mitochondria has been identified [217], this subunit

however extends into the matrix and is therefore not involved in cytosolic stimulation of

SDH. Recently it has become apparent that complex II in plant mitochondria is structurally

different from its mammalian, protist and fungal counterparts. Complex II has been

characterized for bacteria, protozoa, fungi and animals and nearly always is composed of

four subunits [218] : SDH1 to SDH4 (see section 1.2.4). Recently however it was

determined by Eubel and colleagues using 2D Blue-native/SDS PAGE that SDH in potato

mitochondria contains seven subunits [219] of which three were positively identified as

SDH1, SDH2 and SDH3. In follow-up research Millar et al. subsequently found eight

complex II subunits in Arabidopsis thaliana, bean and potato mitochondria [218]. In line

with existing nomenclature the new subunits were named SDH5 to SDH8, apparent

molecular masses being: 65 kDa (SDH1), 29 kDa (SDH2), 18 kDa (SDH5), 15 kDa

(SDH6), 12 kDa (SDH3), 7 kDa (SDH7), 6 kDa (SDH4) and 5 kDa (SDH8). As in other

heterotrophic eukaryotes all subunits are nuclear encoded, i.e. also in plant mitochondria

complex II is the only main ETC complex, which does not contain mitochondrial-encoded

subunits. Sequence comparisons between subunits SDH5 to SDH8 with protein databases

did not allow identification of similar proteins of known functions [218]. Possibly one or

more of the newly identified subunits of the plant SDH face the IMS and contain(s)

phosphorylation sites. The difference in structure may also explain differences in

105

mammalian and plant SDH regulation. Affourtit found that SDH in rat liver mitochondria is

fully activated by succinate alone and does not require addition of ATP (cf. Figure 6B in

[21]). In another study it was found that ATP has no effect on SDH in rat liver

mitochondria as the addition of ATP to rat liver respiring on succinate did not lead to an

increase in , (cf. Figure 1B in [44]). Future research will have to clarify this.

Time dependent effects of ATP addition:

Fricaud et al. determined that potato mitochondria synthesize ATP under ADP limited

conditions [110]. It was found that upon addition of succinate there was a significant

increase in ATP concentration. Experiments showed that endogenous ADP present upon

isolation was used for phosphorylation and the ATP produced was converted back to ADP

by adenylate kinase. This explains the effects of ATP-synthase inhibitors under ADP

limited conditions, is increased and vO2 is decreased because the continuous proton

influx through the ATP synthase is blocked, which results in decreased IMM conductivity.

As discussed in section 1.2.9 ATP synthase activity is regulated by the natural inhibitor

protein IF1. Upon increase of this protein unbinds and ATP synthase activity increases.

When ATP concentration increases (addition of ATP or after a state 3 to state 4 transition)

IF1 is stimulated to bind to the ATP synthase and inhibits it. This was also found with

NADH as a substrate, therefore the effects of added ATP or oligomycin are not specifically

related to SDH activity [110]. Addition of these substances (ATP or oligomycin) has an

inhibitory effect on cytochrome pathway activity, which leads to a reduction in rate b, see

Figure 3.10. This will therefore lead to an increase in Qr/Qt. The addition of ATP early

during the trace will result in stimulation of SDH but at the same time it works as a ‘brake’

on respiration by inhibiting the ATP synthase, as can be seen in Figure 3.14A where

acceleration of vO2 is stopped immediately upon addition of ATP. This ‘brake’ does not

occur in the situation where succinate is the only added substance since higher vO2 rates are

attained in the absence of ATP. Addition of ATP leads to the establishment of a steady state

which is reached instantaneously whereas upon addition of succinate a steady state is only

reached after a relatively long time. When ATP is added after ~6 minutes to potato

mitochondria respiring on succinate, SDH is almost completely activated and SDH has very

106

little control on respiration. It is known that under ADP limited conditions in potato

mitochondria the proton leak has strong control [133]. Inhibiting part of this leak through

the ATP synthase (with either ATP or oligomycin) will have a strong effect. This effect

may be masked early on during the experiment since SDH was not fully activated yet. As

long as SDH is activating the oxygen consumption rate will increase, inhibition of proton

leak during this period of activation may therefore not be apparent. When SDH is fully

activated though; a decrease in oxygen consumption rate due to inhibition of proton leak

can no longer be masked (cf. figures 3.14B, 3.15 and 3.16).

Does uncoupling lead to decreased succinate transport?

Succinate is transported into the matrix via the dicarboxylate carrier which catalyses the

electroneutral exchange of dicarboxylates (e.g. malonate, malate and succinate), inorganic

phosphate and inorganic sulfur containing compounds (e.g. sulfite) [220]. The carrier is

inhibited by butylmalonate [221]. In combination with the phosphate carrier which

catalyses the symport of inorganic phosphate and a proton [43] succinate uptake can be

viewed as a net succinate/proton symport [222], see Figure 3.17.

Figure 3.17 Succinate antiport coupled to phosphate proton symport, the dicarboxylate/phosphate translocase

and the H+:Pi

- symport (equivalent to a OH

-/Pi

- antiport).

succinate Pi

Pi

H+

H+

107

Both transporters catalyse electroneutral processes so dissipation of by uncouplers

should not affect their activity. Uncouplers also dissipate any pH present, which would

decrease the driving force for the ‘succinate/proton symport’ process. However, as

mentioned in section 3.1.2 potato mitochondria contain a highly active K+/H

+ exchanger

which keeps the pH very low. Even though the driving force is small, an effect of

uncoupling on succinate transport in plant mitochondria has been reported [223]. In castor

bean (Ricinus communis) mitochondria dicarboxylate accumulation in the matrix is

inhibited by addition of uncouplers (CCCP and DNP17

) and by inhibitors of the ETC

(antimycin A and KCN). This was interpreted as an effect on phosphate uptake.

Interestingly enough it was found that addition of ATP led to an increase in dicarboxylate

accumulation into the matrix [223]. Which was interpreted as a direct effect on the

dicarboxylate/phosphate translocase. This ATP induced increase in succinate transport into

the matrix was not affected by oligomycin. These findings suggest that dissipation of the

protonmotive force (due to uncoupling or inhibition of the ETC) does not affect SDH

activity directly. Furthermore, a stimulatory effect of ATP on succinate dependent

respiration could be due to an increase in the rate of succinate transport. Oestreicher et al.

used the PMS-DCIP assay (see section 3.1.3) to demonstrate that in plant mitochondria

(cauliflower and mung bean) addition of uncoupler does not deactivate SDH [171] which

suggests that any effects of uncoupling on succinate dependent respiration may be indirect,

e.g. by inhibition of succinate transport.

Is SDH activity regulated by ?

By inhibition of succinate transport the protonmotive force can affect SDH activity, but is

there an additional effect of the protonmotive force in SDH directly? Affourtit reports that

SDH, in the presence of ATP is more active than in the absence of ATP. Figure 3.18 was

taken from [153] (Figure 6.6C). It is readily apparent that in the presence of ATP, at a

certain value of Qr/Qt the respiratory activity is higher with ATP-activated SDH in

comparison to inactive SDH (no ATP present). The ATP-activated data points are in fact

from two data sets, state 3 data () and state 4 () data. The choice of pooling the ATP-

17

carbonyl cyanide m-chlorophenylhydrazone and 2,4-dinitrophenol respectively.

108

activated SDH data points and fitting it with one curve is interesting. This would suggest

that there are no differences in SDH activity depending on protonmotive force, which is the

exact opposite of what Affourtit was trying to demonstrate.

Figure 3.18 Reducing pathway kinetics in potato mitochondria, figure taken from [153], the same data was

used in Figure 5B in [21]. Inactive SDH: reducing pathway kinetics from potato mitochondria respiring on

succinate in the absence of ATP. ATP-activated SDH: reducing pathway kinetics of pooled state 3 () and

state 4 () data. Data modelled according to [76].

The data points from Figure 3.18 were extracted from the plot (a very useful feature of the

Kaleidagraph software package) and new fits were made. This time states 3 and 4 were

treated separately and it can be seen in Figure 3.19 that there is no single curve which can

fit both these data sets (this might explain the reason why the state 3 and state 4 data points

were combined in [21]). In fact, under state 4 conditions, where the magnitude of the

protonmotive force is higher than under state 3 conditions, SDH is less active, i.e. at a

certain value of Qr/Qt the respiratory activity under state 4 conditions is lower than under

state 3 conditions, which is the opposite of what Affourtit hypothesized. In the view of this

author, Figure 5B in [21] if represented as done in Figure 3.19 would have made a very

0

150

300

0.0 0.5 1.0

SĘ

3Ę

T,4Ę

inactive fit

activated fit

V (

nm

ol

O2 m

in-1

mg

pro

tein

-1)

Q-reduction (fraction)

inactive SDH

ATP-activated SDHC

109

strong argument in favour of the hypothesis that SDH activity in potato mitochondria is

indeed modulated by the protonmotive force.

Figure 3.19 Data from Figure 3.18, reducing pathway kinetics under state 3 (red line) and state 4 (green line)

separately modelled according to [76]. values (see section 2.4.5) for state 3 (-0.68) and for state 4 (-0.92).

Linear correlation coefficients (R) for state 3 and state 4 were 0.95143 and 0.64871 respectively.

Still, the data in Figure 3.19 can be explained in terms of an effect on phosphate transport

as opposed to a direct effect of the protonmotive force on SDH. This begs the question

whether or not there are any studies which show conclusively whether or not the

protonmotive force can directly affect SDH activity. The only known example of regulation

of SDH by is in Bacillus subtilis [224]. Complex II in B. subtilis is structurally different

from its eukaryotic counterparts as it has two heme groups [225] and it therefore does not

belong to the same class of SQORs (see section 1.2.4) as the SDH complexes in plant, yeast

or mammalian mitochondria [226]. Complex II in B. subtilis uses menaquinone (MK) as an

electron acceptor. The electron transport from succinate (E° = +30 mV) to MK (E°

-80

mV) [224] is endergonic. It was found that in B. subtilis and other bacteria which use MK

as the respiratory quinone that addition of uncoupler (CCCP) or valinomycin (which

0

150

300

0.0 0.5 1.0

SĘ

3Ę

T,4Ę

inactive fit

activated fit

V (

nm

ol O

2 min

-1 m

g p

rote

in-1

)

Q-reduction (fraction)

inactive SDH

ATP-activated SDH

state 4

state 3

110

dissipates the membrane potential) specifically inhibited succinate dependent respiration

and menaquinone oxidoreductase activity [224]. Upon oxidation of succinate electrons are

transferred ultimately to a heme group which is close to the extra cellular side of the

membrane, i.e. to the positive side. The electron transfer from the heme group close to the

cytosolic side (negative) to the heme group close to the extra cellular side is driven by the

protonmotive force. It was suggested by Schirawski et al. that it is the protonmotive force

which provided the driving force for MK reduction [224]. Although this mechanism allows

for direct stimulation of SDH by the protonmotive force it cannot be used as a means to

modulate SDH activity in eukaryotic mitochondria for the simple reason that complex II in

eukaryotic systems only has one heme group (see figures 1.5 and 1.6). Structural

differences aside, in a later study by Azarkina et al. [227] it was determined that the

stimulating effects of energization on electron transfer in the respiratory chain of B. subtilis

is not associated uniquely with complex II activity, i.e. stimulating effects of energization

were also observed with other substrates and it was hypothesized that electron transfer in

the respiratory chain of B. subtilis might be controlled by membrane energization at the

level of MK reduction by the dehydrogenases. In short, SDH activity in B. subtilis does not

appear to be directly modulated by the protonmotive force. Concluding, given the current

experimental evidence available it seems unlikely that SDH activity is modulated by the

protonmotive force directly, whereas inhibition of succinate transport could explain the

effects of membrane deenergization on succinate dependent respiration.

Summary:

ADP and ATP do not stimulate SDH via . It has recently become apparent that plant

SDH is structurally different from mammalian and yeast SDH, containing at least 8 extra

sub-units with unknown functionality. ADP and ATP probably stimulate SDH

cytosolically, there is no evidence suggesting the presence of phosphorylation sites on

subunits C and D, however phosphorylation sites might be present on one or more of the

newly identified subunits, which still need to be characterised. Findings by Affourtit and

Fricaud appeared to be at variance. It was shown that findings of both Affourtit and Fricaud

could be reproduced in the same system. ATP and oligomycin affect respiratory rates in a

111

substrate independent manner by inhibiting complex V which is continuously active.

Inhibition of the ATP-synthase decreases both IMM conductivity and cytochrome pathway

activity. Decreased cytochrome pathway activity, just like a titration with antimycin, will

result in an increase in Qr/Qt concomitant with a decrease in vO2. When succinate is added

to potato mitochondria SDH slowly becomes activated by succinate itself as it is a

homotropic enzyme. Naturally addition of an activator such as ATP will not stimulate SDH

any further when it is already fully activated, but a decrease in respiration rate would not be

expected. ATP addition has a dual effect. It activates SDH (if it is not fully activated

already by succinate) and simultaneously inhibits cytochrome pathway activity. At the

beginning of an experiment, this inhibitory effect is not apparent because SDH is

spontaneously activating and the net result of addition of ATP will be a net increase in vO2.

Expressing SDH activation in terms of Qr/Qt was an innovative idea which allowed for a

quantitative measure of SDH activation state, determining SDH activity in this way

requires a tightly coupled environment, which turns out to be the Achilles heel of this

approach. In a tightly coupled environment in which the Q-redox poise is determined by the

interaction of Q-reducing and Q-oxidising pathways quantification of enzyme activity in

terms of Qr/Qt is only possible if a change in Qr/Qt is caused only by the enzyme under

investigation, which is not the case when ATP is used as an activator. It is the author’s view

that at present the best way of determining SDH activity is by spectrophotometic

determination of the rate of artificial electron acceptor reduction by SDH.

112

Chapter 4

Respiratory characteristics of Schizosaccharomyces pombe

mitochondria

4.1 INTRODUCTION

The yeast Schizosaccharomyces pombe also referred to as ‘the other yeast’ [95] is

increasingly the preferred model system to investigate a wide range of processes such as the

cell cycle [96], DNA repair [97], microtubule formation, meiotic differentiation, cellular

morphogenesis and stress response mechanisms [98] over the traditionally used

Saccharomyces cerevisiae. S. pombe divides by fission [228] and is one of the few free-

living eukaryotic species whose genome has been completely sequenced at the time of

writing [99]. Although S. pombe has been extensively used to investigate the cell cycle and

genome repair mechanisms, in comparison to S. cerevisiae, relatively little work has been

done on S. pombe metabolism [103] and even less has been done on the respiratory

characteristics of its mitochondria [104, 105]. S. pombe has been used in this laboratory to

express the alternative oxidase (AOX) protein from the plant Sauromatum guttatum in

order to investigate structure function relationships [59]. AOX is a non-protonmotive

terminal oxidase which catalyses the oxidation of ubiquinol (QH2) to ubiquinone (Q) and

the reduction of O2 to H2O [229]. The characteristics of AOX activity and its influence on

overall respiration in S. pombe mitochondria expressing AOX are described in chapters five

and six (see section 1.2.11 for a description of the alternative oxidase). To interpret the

results obtained from work on S. pombe mitochondria expressing the alternative oxidase it

is necessary to have an accurate and complete understanding of the respiratory

characteristics of the ‘wild type’ S. pombe (sp.011 wt) mitochondria. In this study, for the

first time, the membrane potential (∆) in S. pombe mitochondria was measured using the

TPP+ electrode [154]. The presence of a ∆ across the inner mitochondrial membrane of S.

113

pombe mitochondria was already inferred from transport studies done by Moore et al.

[104]. The magnitude of the membrane potential in isolated yeast mitochondria under ADP

limited conditions is in the range of –180 to –200 mV [160, 187], which is intermediate of

what has been reported in mammalian and plant mitochondria (see section 3.1.2) although

lower values have been reported [230]. Characterisation studies on sp.011 wt mitochondria

have been performed previously [26, 104, 105] however was not determined in these

studies. Attempts to optimise the in-house isolation method led to a small improvement in

the quality and yield of isolated mitochondria. Therefore a characterisation of the S. pombe

mitochondria used in this study will be given, presenting values for the membrane

potential, under different energetic conditions, for the first time. Under certain energetic

conditions cytochrome pathway kinetics were obtained which appeared to be different from

what has been seen previously in mitochondria from other sources. Cytochrome pathway

kinetics in sp.011 wt mitochondria were investigated extensively and comparisons were

made with cytochrome pathway kinetics from potato and Saccharomyces cerevisiae

mitochondria.

4.2 RESULTS

4.2.1 General characterisation of Schizosaccharomyces pombe respiratory

kinetics

4.2.1.1 Respiratory rates with different substrates—Table 4.1A shows averaged oxygen

consumption rates (vO2) under different energetic conditions. Concentrations of added

chemicals, the number of data points and the amount of mitochondrial isolations are given

in the legend.

NADH-dependent respiration:

Under state 2 conditions the average vO2 was 91 nmol O2 / min / mg protein. Upon addition

of a limited amount of ADP (0.2 mM) the averaged vO2 increased almost three times to 281

nmol O2 / min / mg protein (state 3), which demonstrates that electron transfer is coupled to

114

phosphorylation. Upon depletion of ADP (state 4) the average vO2 decreased to 110 nmol

O2 / min / mg protein. Upon addition of an uncoupler (CCCP, 2 M) the vO2 increased

almost six times compared to the state 2 rate, which indicates that the mitochondria upon

isolation are relatively intact. The IMM functions as an isolating membrane, which is a

requirement for an energy transducing system (see postulate 4 on page 35). The state 3 rate

/ state 4 rate RCR is 2.6 and the uncoupled / state 2 RCR is 5.9 (Table 4.1B).

Succinate-dependent respiration:

Addition of succinate alone led to an averaged vO2 of 57 nmol O2 / min / mg protein which

is considerably lower than the NADH state 2 rate. Comparable to plant species, SDH needs

to be activated [21, 216]. Successive additions of ATP [123] and glutamate [104] (for

removal of oxaloacetate) led to further increases in vO2. The mechanism by which ATP

activates SDH is still poorly understood [21] but possibly ATP interacts directly with SDH

cytosolically (see chapter 3). Unlike in potato mitochondria the effect of adding ATP to S.

pombe mitochondria is modest, only small increases in Qr/Qt and are seen (cf. figures

3.5 and 3.7 respectively) a possible explanation for this difference between plant and yeast

mitochondria is that apart from stimulating complex II [123] ATP has been found to

stimulate complex IV in yeast mitochondria [230]. Stimulation of an oxidising pathway

will lead to a decrease in steady state Qr/Qt.

Oxaloacetate is known to inhibit SDH when bound to it, addition of glutamate

results in transamination of oxaloacetate to aspartate [231]. A decrease in protonmotive

force (p) is known to lead to a decrease of SDH activity [21, 26]. Uncoupled rates (144

nmol O2 / min / mg protein) are lower than state 3 rates (156 nmol O2 / min / mg protein)

which suggests that SDH is deactivated due to dissipation of p. Deactivation of SDH is

also reflected in the succinate RCR values. For succinate the state 3 / state 4 RCR value is

1.6 and the uncoupled / state 2 RCR value is 1.4 (Table 4.1B). Deactivation of the

dehydrogenases will be discussed in section 4.2.3.

115

Glutamate-dependent respiration:

Addition of glutamate itself induces a rate (32 nmol O2 / min / mg protein) which can be

increased further by addition of ATP (43 nmol O2 / min / mg protein). This has been

interpreted as SDH activation by Crichton [26]. He hypothesizes that addition of glutamate

leads to generation of succinate inside the matrix. This hypothesis is supported by findings

in this study. Addition of glutamate led to a slowly accelerating vO2, as seen when

succinate is added as a substrate (cf. Figure 3.13), which suggests that SDH is slowly

activating. Addition of ATP led to an increase in vO2 which can be interpreted as activation

of SDH by ATP [21]. Addition of ADP or CCCP did not lead to an appreciable increase in

rate, which can be interpreted as deactivation of SDH. Most convincingly though is the

observation that upon addition of malonate, an inhibitor of complex II (see section 3.1.3)

the glutamate-dependent respiration is completely inhibited (data not shown).

The glutamate-dependent respiratory rate (in the presence of ATP) is lower than the

succinate-dependent respiratory rate. This could be due to SDH not being fully activated

when the concentration of succinate generated after glutamate addition is subsaturating.

Possibly the addition of glutamate leads to release of a limited amount of oxaloacetate,

which then (via the TCA cycle) is converted to succinate. If indeed succinate concentration

would be subsaturating then the membrane potential with glutamate as a substrate should

be lower than with succinate as a substrate. This would be the basis for an interesting

experiment in future work. S. pombe mitochondria contain glutamate dehydrogenase

(SPCC132.04c)18

which can couple the oxidation of glutamate to -oxoglutarate to the

reduction of NAD+ thereby generating matrix NADH [43]. This could also lead to

glutamate dependent respiration but given the results obtained with malonate this seems

unlikely.

Table 4C shows the ADP/O ratios obtained with NADH or succinate as a substrate

being 1.36 and 1.22 respectively. The values are somewhat low. S. pombe mitochondria do

not express complex I therefore only complexes III and IV translocate protons across the

IMM. The expected ADP/O value for either NADH or succinate would be 1.5 [1]. Poor

phosphorylative capacity may reflect the in vivo role of mitochondria in S. pombe.

18

http://www.genedb.org/genedb/Search?organism=pombe&name=SPCC132.04c&isid=true

116

Table 4D shows the sensitivity of sp.011 wt mitochondria to inhibitors. KCN, antimycin A

and myxothiazol completely inhibit NADH and succinate dependent respiration which

indicates that no alternative oxidase is expressed in S. pombe mitochondria as reported

previously [104]. Octyl gallate, a potent inhibitor of AOX [70] at a concentration of 16 µM

slightly inhibits both NADH-and succinate-dependent respiration. SDH can be fully

inhibited by malonate. Carboxyattractyloside (CAT) is a potent inhibitor of the adenine

nucleotide translocator [126], addition of 5 L of a 1 mg/ml solution of CAT completely

prevents generation of a state 3 upon addition of an aliquot of ADP.

Table 4 E shows averaged membrane potentials obtained with NADH or succinate

(ATP and glutamate incubated). The obtained ∆ values with either substrate are

approximately the same, indicating that, with respect to the membrane potential there are

no substrate dependent differences. Under state 4 conditions with either substrate the

magnitude of ∆ is smaller than under state 2 conditions. The increased vO2 and decreased

∆ under state 4 conditions are suggestive of an increased conduction of the IMM. These

observations may indicate the involvement of adenylate kinase (see section 1.3.1). This

enzyme catalyses the reaction: ATP+AMP 2 ADP [110]. With each state 3 more ATP

will be available for ADP recycling, leading to increased ATP-synthase activity. The

presence of adenylate kinase in S. pombe mitochondria has been demonstrated [112]. Yeast

AK is located in the IMS where it can move freely, unlike in plants where it is bound to the

outer face of the IMM [232].

117

A Respiratory Rate (nmol O2 min-1

mg-1

protein)

substrate(s) state 2 state 3 state 4 uncoupled

NADH 91 (13) 281 (39) 110 (22) 537 (68)

succinate * 106 (15) 156 (18) 97 (7) 144 (25)

glutamate 32 (4) - - -

glutamate + ATP 43 (8) - - -

B RCR

substrate state 3 / state 4 uncoupled / state 2

NADH 2.6 5.9

succinate *

1.6 1.4

C ADP/O

NADH 1.36 (0.13) [20,11]

succinate * 1.22 (0.08) [8,4]

D Sensitivity to inhibitors

inhibitor percentage of oxygen consumption rate inhibition

antimycin A (2.3 µM) 100%

KCN (270 µM) 100%

myxothiazol (2.5 L of 1 mg/ml) 100%

malonate (9 mM) 100%

octyl gallate (16 M) 10%

E ∆ values (mV)

substrate state 2 state 3 state 4

NADH 208 (6) [6,4] 188 (7) [4,2] 204 (11) [4,2] **

succinate *

200 (7) [4,4] 180 (10) [3,2] 195 (5) [3,2] **

Table 4.1 Respiratory characteristics of sp.011 wt mitochondria.

Table A: respiration rate ( S.D.) (see Appendix 2A for full details regarding amount of mitochondrial

isolations and experimental traces performed). Concentrations of chemicals added: NADH (1.8 mM),

succinate (9 mM), ATP (0.2 mM), glutamate (9 mM). Uncoupled conditions are defined as respiratory

118

activity in the presence of an uncoupler (2 µM CCCP). The concentration of added ADP to induce a state 3 to

state 4 transition was 0.2 mM in all cases. State 3 conditions are defined as respiratory activity in the presence

of 1 mM ADP. The RCR values (see section 2.4.4.3) in table B were calculated using the vO2 values from

Table 4.1A. Table C: ADP/O value ( S.D.) [traces, preps]. Table D: percentage inhibition by inhibitors.

Table E: values ( S.D.) [traces, preps]. Nomenclature used is discussed in section 2.4.4.2.

* SDH is activated stepwise by addition of ATP (0.2 mM) and glutamate (9 mM). vO2 with just succinate is

57 (7) addition of ATP subsequently leads to a vO2 of 72 (12).

** ∆ for the first state 4 only

Figure 4.1 shows a representative trace of an experiment where ∆ and vO2 were measured

simultaneously. Upon addition of NADH (1.8 mM) a membrane potential of 210 mV was

generated with a concomitant vO2 of 120 nmol O2 / min / mg protein. Upon addition of

ADP (0.2 mM) ∆ was lowered to 190 mV and the respiratory rate increased to 287 nmol

O2 / min / mg protein, which indicates that electron transfer is coupled to phosphorylation.

After depletion of ADP stabilised at a value of 203 mV with a concomitant vO2 of 110

nmol O2 / min / mg protein. Upon addition of CCCP (2 M) was dissipated and vO2

increased to 630 nmol O2 / min / mg protein. These findings are in agreement with what has

been found previously in mitochondria of S. cerevisiae [160], plants [157] and mammals

[1].

The protonmotive force (pmf) in mitochondria is composed of an electrical potential

(∆) and a proton concentration gradient (∆pH) [1]. Nigericin is an ionophore which can

bind both H+

or K+, this molecule, when added to a solution of mitochondria, will catalyse

the electroneutral exchange of H+ for K

+, which leads to dissipation of ∆pH [1]. After

addition of nigericin the pH will be converted to . Addition of nigericin to S. pombe

mitochondria respiring on either NADH or succinate did not lead to a change in TPP+-

electrode signal (data not shown), indicating that as in potato mitochondria [157] the ∆pH

component of the pmf in S. pombe mitochondria is negligible. The absence of a detectable

∆pH suggests the presence of a K+/H

+ exchanger [188] in S. pombe mitochondria.

Figure 4.2 shows representative oxygen consumption and traces from sp.011

wt mitochondria respiring on succinate. Addition of succinate (9 mM) generated a

membrane potential of 193 mV with a low concomitant vO2 of 39 nmol O2 / min / mg

protein. Addition of ATP (0.2 mM) stimulated SDH as seen in the increase of vO2 to 64

119

nmol O2 / min / mg protein, however was seen to decrease slightly (using a fresh ATP

stock), addition of glutamate (9 mM) led to further increases in both vO2 and , to 96

nmol O2 / min / mg protein and 198 mV respectively. Addition of ADP led to a decrease in

to 179 mV and an increase in vO2 to 138 nmol O2 / min / mg protein, which indicates

that respiration is coupled to phosphorylation, but not to the same extent as with NADH.

This can be interpreted as deactivation of SDH. After depletion of ADP vO2 decreased to

109 nmol O2 / min / mg protein and increased to 197 mV (state 4). Addition of ADP

(0.2 mM) resulted in a second state 3 to state 4 transition.

Figure 4.1 State 3 to state 4 transition in NADH respiring sp.011 wt mitochondria. Simultaneous

measurement of oxygen uptake (—) and membrane potential (). 1.8 mM NADH, 0.2 mM ADP, 2 M

CCCP. The numbers adjacent to the respiratory traces represent specific O2-uptake rates (nmol O2 / min / mg

protein, bold font) and ∆ values (mV, standard font). The traces shown are representative of data from

repeated experiments. Amount of mitochondrial protein used was 0.5 mg.

0

100

200

300

400

500

600

0.85

0.9

0.95

1

1.05

1.1

1.15

0 1 2 3 4 5 6

ox

yg

en (

nm

ol)

TP

P+ sig

na

l V

time (min)

120

287

110

630

210203

184

NADH ADP CCCP anoxia

120

Figure 4.2 sp.011 wt mitochondria respiring on succinate. Simultaneous measurement of oxygen uptake (—)

and membrane potential (). 9 mM succinate, 0.2 mM ATP, 9 mM glutamate, 0.2 mM ADP, 0.2 mM ADP.

The numbers adjacent to the respiratory traces represent specific O2-uptake rates (nmol O2 min-1

mg-1

) and

∆ values (mV). The traces shown are representative of data from repeated experiments. Amount of

mitochondrial protein used was 1 mg.

Figure 4.3 shows a representative trace of an experiment where Qr/Qt and oxygen

consumption were measured simultaneously in sp.011 wt mitochondria respiring on

succinate19

. Addition of succinate (9 mM) resulted in an artifactual signal followed by

slow SDH activation until a steady state Qr/Qt of 63% was reached with a concomitant vO2

of 56 nmol O2 / min / mg protein. Addition of ATP (0.2 mM) and glutamate (9 mM) led to

further activation of SDH as reflected in increased Qr/Qt and vO2 values to 89% and 95

nmol O2 / min / mg protein respectively. Addition of ADP (0.2 mM) led to an increase in

vO2 to 153 nmol O2 / min / mg protein and a decrease in Qr/Qt to 66% which indicates that

respiration is coupled to phosphorylation. Upon depletion of ADP a state 4 was reached

with Qr/Qt 92% and vO2 95 nmol O2 / min / mg protein. Another addition of ADP (0.2) led

to a state 3 which coincided with anoxia where the Q signal stabilised at 100% Qr/Qt. The

data shown in Figure 4.3 agree well with results previously obtained in this laboratory [26].

19

An equivalent Qr/Qt trace with NADH cannot be produced because of the strong interference of NADH

with the Q-electrode at high concentrations (1.8 mM).

1.2

1.25

1.3

1.35

1.4

1.45

1.5

1.55

1.6

0

100

200

300

400

500

600

0 5 10 15 20

TP

P+ s

ign

al

(V) o

xy

gen

(nm

ol)

time (min)

succinate ATP glutamate ADPADP anoxia

64

96

138

39

109

152

107

193189

198

179

197

175

193

121

Figure 4.3 Effects of ATP, glutamate and ADP on succinate-dependent respiratory activity. Simultaneous

measurement of oxygen uptake () and redox poise of the mitochondrial Q-pool (…) in sp.011 wt

mitochondria respiring on succinate. 9 mM succinate, 0.2 mM ATP and 9 mM glutamate, 50 M ADP and

0.1 mM ADP were added where indicated. The numbers adjacent to the respiratory traces represent specific

O2-uptake rates (nmol O2 / min / mg

protein) and Q-reduction levels (fraction QH2 in total pool). The traces

shown are representative of data from repeated experiments. Amount of mitochondrial protein used was 0.7

mg.

The sp.011 wt mitochondria isolated in this study were shown to be of good quality. With

NADH as a substrate the RCR value (uncoupled / state 2) was ~5.9 which indicates that the

IMM is relatively impermeable to protons (see postulate 4 on page 35). Respiration rates of

sp.011 wt mitochondria respiring on succinate become limited under state 3 and uncoupled

conditions which suggests deactivation of SDH upon a decrease in . The ability of these

mitochondria to phosphorylate ADP is relatively poor with respect to what is seen in

mammalian and plant mitochondria. The membrane potential generated and its response to

changes in energetic conditions in sp.011 wt mitochondria is similar to what is seen in

mitochondria isolated from S. cerevisiae [160], plants [157] (cf. Figure 3.2) and mammals

[1].

0

100

200

300

400

500

600

0

0.2

0.4

0.6

0.8

1

0 2 4 6 8 10 12

ox

yg

en (

nm

ol)

Qr/Q

t

time (min)

succinate

ATP

glutamate

ADP

ADP

anoxia

0.63 0.64

0.89

0.66

0.92

0.66

56

67

95

153

95

150

122

4.2.2 Schizosaccharomyces pombe - cytochrome pathway kinetics

Cytochrome pathway kinetics (Qr/Qt vs. vO2) have been determined previously in both

sp.011 wt mitochondria [104] and S. pombe mitochondria transformed with AOX [93], by

titrating succinate-dependent respiration with malonate. Cytochrome pathway kinetics with

NADH as a substrate were not determined in previous studies as NADH interacts with the

Q-electrode. The NADH regenerating method (with Glucose-6-Phosphate dehydrogenase

and Glucose-6-Phosphate incubated), in which subsaturating amounts of NADH are added

to activate the external NADH dehydrogenase, can be used effectively, since additions of

small amounts of NADH lead to only small effects on the Q-signal which can be corrected

for (see section 2.4.4.4). Oxidising pathway kinetics of S. pombe mitochondria with NADH

as a substrate have not been determined before. As part of a full characterisation of the

sp.011 wt mitochondria it was decided to determine cytochrome pathway kinetics under

various energetic conditions (states 2 to 4 and uncoupled) with NADH as a substrate

measuring Qr/Qt, vO2 and ∆ simultaneously or in parallel.

To obtain state 3 or uncoupled oxidising pathway kinetics; a saturating amount of

ADP (1 mM) or a saturating amount of CCCP (2 M) was incubated. To obtain state 4

oxidising pathway kinetics a state 3 to state 4 transition was induced by adding an

aliquot of ADP (~50 M).

4.2.2.1 The relationships of Qr/Qt vs. vO2 and ∆ vs. vO2 under ADP limited

conditions with NADH as a substrate—Figure 4.4 shows the relationship between Qr/Qt

and vO2 under state 2 and state 4 conditions. Under state 2 conditions no ADP is added (see

section 2.4.4.2). State 4 conditions were achieved by titrating with subsaturating amounts

of NADH until a low rate of vO2 was achieved followed by the addition of an aliquot of

ADP (~50 µM) which led to a transient increase in vO2 and concomitant decrease in Qr/Qt,

upon depletion of ADP the NADH titration was continued and only those data points were

used to represent state 4 cytochrome pathway kinetics.

123

0

20

40

60

80

100

120

0 0.2 0.4 0.6 0.8 1

vO

2 n

mol

O2 /

min

/ m

g p

ro

tein

Qr/Qt

0

10

20

30

40

50

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

rote

in

Qr/Qt

A

Figure 4.4 Relationship between Qr/Qt and vO2 under ADP limited conditions with NADH as a substrate.

Using the NADH regenerating system Qr/Qt and vO2 were measured simultaneously under state 2 () and

state 4 conditions (). State 4 conditions were achieved by titrating with subsaturating amounts of NADH (~

3-5 M) until a low rate of vO2 was achieved followed by the addition of an aliquot of ADP (typically 50

µM) which led to a transient increase in vO2 and concomitant decrease in Qr/Qt, upon depletion of ADP the

NADH titration was continued. State 2 and state 4 data points overlap and the relationship between Qr/Qt and

vO2 is linear. For comparison the same relationship under state 4 conditions in fresh potato mitochondria,

obtained with the NADH regenerating system is shown in inset A. This relationship is linear up until high

values of Qr/Qt where its shape becomes slightly convex. The sp.011 state 2 and state 4 titration data were

obtained from six and seven mitochondrial isolations respectively, data points were combined from nine and

ten traces respectively. The state 4 potato titration data was obtained from one mitochondrial isolation

combining data from two traces. Mitochondrial protein used per experiment for S. pombe (0.5-0.7 mg) and for

potato (0.5-1 mg).

124

The data points from both conditions overlap and the relationship between Qr/Qt vs. vO2 is

linear. As a comparison, in the inset, the relationship between Qr/Qt and vO2 is shown in

fresh potato mitochondria under state 4 conditions (state 4 conditions were achieved in the

same way as for S. pombe mitochondria), which is primarily linear becoming slightly

convex at high rates. Linear relationships (Qr/Qt vs. vO2) are typically seen under ADP

limited conditions in mitochondria which do not have a branched respiratory chain [76, 86].

A linear relationship between Qr/Qt and vO2 under ADP limited conditions has been

demonstrated previously in S. pombe mitochondria respiring on succinate (cf. Figure 2 in

[104]), this suggests that there are no substrate dependent differences in cytochrome

pathway kinetics under ADP limited conditions. These results agree well with data

previously obtained in this laboratory and with those reported in the literature.

Figure 4.5 shows the relationship between ∆ and vO2 under state 2 and state 4

conditions in sp.011 wt mitochondria. Data from both conditions overlap and it can be seen

that under state 4 conditions higher respiratory rates are obtained, possibly reflecting the

activity of adenylate kinase. Immediately apparent from the data is a non-ohmic

relationship between ∆ and vO2 (see section 3.1.2). A non-ohmic p vs. proton

conductance relationship under ADP limited conditions has been shown previously in S.

cerevisiae mitochondria [233]. The results presented here agree well with the yeast

literature. As a comparison the same relationship under state 4 conditions as determined in

fresh potato mitochondria is given (data points taken from Figure 3.4).

4.2.2.2 The relationships of Qr/Qt vs. vO2 and ∆ vs. vO2 under state 3 conditions with

NADH as a substrate—Figure 4.6 shows the relationship between Qr/Qt and vO2 under

state 3 conditions in sp.011 wt mitochondria. It is apparent that the relationship is non-

linear, showing a biphasic shape. This was surprising since in other tissues this relationship

under state 3 conditions normally is found to be linear [37, 234], the inset shows the

relationship in fresh potato mitochondria. At low Qr/Qt there is little respiratory activity,

which increases considerably between 30 and 40 % Q-reduction in the sp.011 wt

mitochondria.

125

0

50

100

150

100 120 140 160 180 200 220

vO

2 n

mo

l O

2 /

min

/ m

g p

ro

tein

membrane potential (mV)

Figure 4.5 The relationship between ∆ and vO2 under state 2 and state 4 conditions with NADH as a

substrate. Using the NADH regenerating system both vO2 and ∆ were measured simultaneously under state

2 ( ) and state 4 () conditions. State 4 conditions were achieved by titrating with subsaturating amounts of

NADH (~ 3-5 M) until a low rate of vO2 was achieved followed by the addition of an aliquot of ADP (50

M) which led to a transient increase in vO2, upon depletion of ADP the NADH titration was continued. The

relationship between ∆ and vO2 under both state 2 and state 4 conditions is non-ohmic. For comparison the

same relationship was determined in fresh potato mitochondria under state 4 conditions using the NADH

regenerating system. The sp.011 state 2 and state 4 titration data were obtained from four and two

mitochondrial isolations respectively, data points were combined from six and three traces respectively. The

fresh potato titration data was taken from Figure 3.4. As already seen in Table 4.1A, the vO2 rates under state

4 conditions are higher than under state 2 conditions, possibly indicating adenylate kinase activity in S. pombe

mitochondria. Mitochondrial protein used per experiment for S. pombe mitochondria (0.5-0.7 mg).

0

10

20

30

40

50

60

70

80

170 180 190 200 210 220

vO

2 n

mo

l O

2 /

min

/ m

g p

rote

in


126

0

50

100

150

200

250

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

ro

tein

Qr/Qt

0

20

40

60

80

100

120

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

ro

tein

Qr/Qt

A

Figure 4.6 The relationship between Qr/Qt and vO2 under state 3 conditions with NADH as a substrate in

sp.011 wt mitochondria. Using the NADH regenerating system both vO2 and Qr/Qt were measured

simultaneously under state 3 conditions. Mitochondria were incubated with ADP (1 mM). The relationship

between Qr/Qt and vO2 shows a biphasic pattern, a change in slope is observed at around 30 to 40 % Q-

reduction. For comparison, the same relationship in fresh potato mitochondria under state 3 conditions (ADP

1 mM), obtained using the NADH regenerating system, is shown in inset A. In potato mitochondria a slight

convex shape at high Qr/Qt is observed reminiscent of the pattern seen under state 4 conditions (see Figure

4.5 inset A).The sp.011 wt titration data were obtained from five mitochondrial preparations, combining data

from eleven traces. The potato titration data were obtained from one mitochondrial isolation, combining data

from two traces. Mitochondrial protein used per experiment for S. pombe (0.5-0.7 mg) and for potato (0.5-1

mg).

127

Figure 4.7 shows the relationship between ∆ and vO2 under state 3 conditions in sp.011

wt mitochondria. A biphasic pattern is again seen in this relationship, and is different from

what is seen in other tissues, where this relationship normally is linear [157], see inset A

where the same relationship is given for fresh potato mitochondria. A representative trace is

given in inset B clearly showing the biphasic nature of the kinetics in sp.011 wt

mitochondria under state 3 conditions. By combined plotting of ∆, Qr/Qt and vO2 it

became clear that the vO2 rate ( 50 nmol O2 / min / mg protein) at which the relationships

of both Qr/Qt vs. vO2 and ∆ vs. vO2 become biphasic is the same, indicating that the

change in both relationships occur simultaneously (data not shown).

4.2.2.3 The relationship between Qr/Qt vs. vO2 under uncoupled conditions with NADH as

a substrate—Since ∆ is dissipated by CCCP only the relationship between Qr/Qt and

vO2 was investigated. Figure 4.8 shows the relationship between Qr/Qt vs. vO2 under

uncoupled conditions in sp.011 wt mitochondria. The relationship becomes biphasic around

40% Q-reduction, for comparison, the inset shows this relationship in fresh potato

mitochondria. The vO2 rate at which the relationship becomes biphasic is the same as

under state 3 conditions (around 50 nmol O2 / min / mg protein) also, the biphasic pattern is

present whilst ∆ is dissipated which suggests that ∆ probably is not instrumental in

causing the biphasic pattern.

Are the biphasic cytochrome pathway kinetics NADH dependent?

In previous work, using succinate as a substrate, the relationships between Qr/Qt and vO2

under state 3 and uncoupled conditions were determined in transformed S. pombe

mitochondria in which AOX expression was repressed, i.e. mitochondria which only use

the cytochrome pathway to oxidise the Q-pool [93]. The biphasic patterns as seen in figures

4.6 and 4.8 were not observed in that study and this raised the question whether or not the

biphasic cytochrome pathway kinetics were NADH-dependent. Therefore cytochrome

pathway kinetics under various energetic conditions (states 2 to 4 and uncoupled) were

determined by titrating succinate-dependent respiration in the presence of malonate.

128

0

50

100

150

200

250

300

350

400

120 130 140 150 160 170 180

vO

2 n

mo

l O

2 /

min

/ m

g p

ro

tein


0

20

40

60

80

100

162 164 166 168 170 172 174

vO

2 n

mo

l O

2 /

min

/ m

g p

ro

tein


A

0

20

40

60

80

100

120

140

160

130 140 150 160 170 180

vO

2 n

mo

l O

2 /

min

/ m

g p

ro

tein


B

Figure 4.7 The relationship between ∆ and vO2 under state 3 conditions in sp.011 wt mitochondria

respiring on NADH. Using the NADH regenerating system, ∆ and vO2 were determined simultaneously

under state 3 conditions. ADP was incubated at a concentration of 1 mM. A biphasic pattern similar to the one

seen in the relationship between Qr/Qt and vO2 (see Figure 4.6) is seen. For comparison the same relationship

under state 3 conditions (1 mM ADP pre-incubated) obtained with the NADH regenerating system was

determined in fresh potato mitochondria (inset A). Due to variability in the sp.011 data the presence of a

biphasic pattern may not be apparent, therefore a representative individual trace is shown in inset B. It is clear

that the relationship between ∆ and vO2 is linear in potato mitochondria whereas it is biphasic in S. pombe

mitochondria. The sp.011 titration data were obtained from three mitochondrial isolations, combined data

from five traces. The potato titration data was from one mitochondrial isolation, data from two traces.

Mitochondrial protein used per experiment for S. pombe (0.5-0.7 mg) and for potato (0.5-1 mg).

129

0

50

100

150

200

250

300

350

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

ro

tein

Qr/Qt

0

20

40

60

80

100

120

140

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

ro

tein

Qr/Qt

A

Figure 4.8 The relationship between Qr/Qt and vO2 under uncoupled conditions in sp.011 wt mitochondria

respiring on NADH. Using the NADH regenerating system both vO2 and ∆ were measured simultaneously

under uncoupled conditions. Mitochondria were incubated with CCCP (2 M). The relationship between

Qr/Qt and vO2 shows a biphasic pattern, a clear change in slope is observed at around 40 % Q-reduction. For

comparison the same relationship in fresh potato mitochondria under uncoupled conditions, obtained by using

the NADH regenerating system, is shown in the inset. A slight convex shape is observed in potato

mitochondria. The sp.011 titration data were obtained from five mitochondrial preparations, combining data

from five traces. The potato titration data were obtained from one mitochondrial isolation, combining data

from three traces. Mitochondrial protein used per experiment for S. pombe (0.5-0.7 mg) and for potato (0.5-1

mg).

130

0

20

40

60

80

100

120

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

ro

tein

Qr/Qt

4.2.2.4 A comparison of NADH and succinate dependent Qr/Qt vs. vO2 and ∆ vs. vO2

relationships under ADP limited conditions—Figure 4.9 shows combined succinate and

NADH Qr/Qt vs. vO2 data which clearly show that under ADP limited conditions there are

no substrate-dependent differences in cytochrome pathway kinetics.

Figure 4.9 Combined state 2 and state 4 cytochrome pathway kinetics of sp.011 wt mitochondria respiring on

either NADH or succinate, Qr/Qt vs. vO2. NADH state 2 () and state 4 () data were taken from Figure 4.4.

Succinate state 2 () and state 4 (∆) data were obtained with the malonate titration method. With succinate as

a substrate a state 4 was induced by adding an aliquot of ADP (50M) after the mitochondria were energised

with succinate (9 mM) + ATP (0.1 mM) + glutamate (9 mM), upon depletion of ADP respiratory activity was

titrated with malonate (up to ~ 4 mM). State 2 and state 4 succinate titration data were obtained from one and

two mitochondrial isolations respectively. Data points for succinate state 2 and state 4 were taken from one

trace and nine traces respectively. Mitochondrial protein used per experiment was 0.5-0.7 mg.

Figure 4.10 shows combined succinate and NADH vs. vO2 data which clearly shows

that under ADP limited conditions there are no substrate dependent differences in kinetics.

It is clear that the relationship between and vO2 is non-ohmic (cf. figures 3.1 and 3.4).

131

0

50

100

150

100 120 140 160 180 200 220

vO

2 n

mo

l O

2 /

min

/ m

g p

rote

in


Figure 4.10 Combined state 2 and state 4 cytochrome pathway kinetics of sp.011 wt mitochondria respiring

on either NADH or succinate, ∆ vs. vO2. NADH state 2 () and state 4 (∆) data were taken from Figure

4.5. Succinate state 2 () were obtained with the malonate titration method (0 – 4 mM malonate). Succinate

titration data were obtained from one mitochondrial isolation, data points were taken from one trace.

Mitochondrial protein used per experiment was 0.5-0.7 mg.

4.2.2.5 A comparison of NADH and succinate dependent Qr/Qt vs. vO2 relationships under

state 3 conditions—Figure 4.11 shows combined succinate and NADH Qr/Qt vs. vO2 data

which show that under state 3 conditions there are no substrate dependent differences in

cytochrome pathway kinetics. The succinate data points overlap with the NADH data

points up until the point where vO2 becomes limiting, this reflects the lower activity of the

SDH in comparison to the external NADH dehydrogenase.

132

0

50

100

150

200

250

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

ro

tein

Qr/Qt

Figure 4.11 Combined state 3 cytochrome pathway kinetics of sp.011 wt mitochondria respiring on either

NADH or succinate, Qr/Qt vs. vO2. NADH data () were taken from Figure 4.6. Succinate data () were

obtained with the malonate titration method (0 – 4 mM malonate). A state 3 was brought about by incubating

with ADP (1 mM). Succinate titration data were obtained from two mitochondrial isolations, data points were

taken from five traces. Mitochondrial protein used per experiment was 0.5-0.7 mg.

4.2.2.6 A comparison of NADH and succinate dependent Qr/Qt vs. vO2 relationships under

uncoupled conditions—Figure 4.12 shows combined succinate and NADH Qr/Qt vs. vO2

data that clearly show that under uncoupled conditions there are no substrate dependent

differences in cytochrome pathway kinetics. The succinate data points overlap with the

NADH data points up until the point where vO2 becomes limiting which, as under state 3

conditions, reflects the lower activity of the SDH in comparison to the external NADH

dehydrogenase.

133

0

50

100

150

200

250

300

350

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

rote

in

Qr/Qt

Figure 4.12 Combined uncoupled cytochrome pathway kinetics of sp.011 wt mitochondria respiring on either

NADH or succinate, Qr/Qt vs. vO2. NADH data () were taken from Figure 4.8. Succinate data () were

obtained with the malonate titration method (0 – 4 mM malonate). Mitochondria were incubated with CCCP

(2 M). Succinate titration data were obtained from five mitochondrial isolations, data points were taken from

eleven traces. Mitochondrial protein used per experiment was 0.5-0.7 mg.

From the data presented in figures 4.9 to 4.12 it was concluded that there were no substrate

dependent differences in sp.011 wt cytochrome pathway kinetics. To further characterize

the S. pombe respiratory kinetics, the reducing pathway kinetics with either NADH or

succinate as a substrate were determined.

134

4.2.3 Schizosaccharomyces pombe - reducing pathway kinetics

Reducing pathway kinetics from S. pombe mitochondria expressing AOX have been

determined previously under state 4 conditions both with either succinate or NADH as a

substrate by titrating respiration with antimycin A [93]. Under ADP limited conditions

is relatively stable, i.e. a large decrease in respiration is concomitant with only a small

decrease in (see section 3.1.2). According to Affourtit an effect of the protonmotive

force on reducing pathway kinetics in S. pombe mitochondria was not expected and

reducing pathway kinetics were not determined under state 3 or uncoupled conditions [93].

It is however known from literature [21, 93] and from findings during this thesis work that

SDH is deactivated upon dissipation of ∆p which led this author to expect that

protonmotive force does have an effect on reducing pathway kinetics with succinate as a

substrate in sp.011 wt mitochondria.

4.2.3.1 SDH reducing pathway kinetics in sp.011 wt mitochondria under state 2 and

uncoupled conditions—Figure 4.13 shows combined SDH reducing pathway kinetics under

state 2 and uncoupled conditions. A comparison between state 2 and uncoupled kinetic data

should reveal clear differences if were to have an effect on SDH activity. Deactivation

of SDH is clearly reflected in the lower slope of the uncoupled data points. Under state 2

conditions, at every value of Qr/Qt the respiration rate is higher when compared to

uncoupled conditions. This indicates that SDH in the presence of is more active than in

the absence of .

The reducing pathway kinetics were determined by titrating respiration with

antimycin A. It has been reported previously that S. cerevisiae mitochondria display linear

relationships between bc1 complex inhibition and respiratory rate [27]. It was suggested

that yeast mitochondria did not display pool behaviour. Figure 4.14 shows a representative

trace of an antimycin A titration in sp.011 wt mitochondria respiring on succinate. A

sigmoidal response can be seen which indicates that S. pombe mitochondria display pool

behaviour [24] (see also section 1.2.5)

135

0

50

100

150

200

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

rote

in

Qr/Qt

Figure 4.13 Combined state 2 and uncoupled reducing pathway kinetics with succinate as a substrate, Qr/Qt

vs. vO2. Reducing pathway kinetics under state 2 () and uncoupled conditions () were determined by

titrating with antimycin A (0 – 40 nM), Qr/Qt and vO2 were measured simultaneously. SDH deactivation is

reflected in the difference between state 2 and uncoupled reducing pathway kinetics. Additions: succinate 9

mM, ATP 0.2 mM, glutamate 9 mM and under uncoupled conditions CCCP 2 M. Mitochondrial protein

used per experiment was 0.5-0.7 mg.

Figure 4.14 Antimycin A titration of sp.011 wt mitochondria respiring on succinate, data taken from Figure

4.13.

0

40

80

120

160

0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1

vO

2 n

mo

l O

2 /

min

/ m

g p

rote

in

Qr/Qt

136

0

10

20

30

40

50

60

70

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

rote

in

Qr/Qt

4.2.3.2 External NADH dehydrogenase reducing pathway kinetics in sp.011 wt

mitochondria under state 2 and uncoupled conditions—Figure 4.15 shows combined

external NADH dehydrogenase reducing pathway kinetics under state 2 and uncoupled

conditions. Respiration was initiated by titrating with sub-saturating amounts of NADH.

Titration was stopped before the external NADH dehydrogenase was fully active in order to

have a large range of Qr/Qt to titrate. When the external NADH dehydrogenase is fully

active the Q-pool is highly reduced (cf. figures 4.4 and 4.8). Titration with antimycin A

then would result in only a few data points from which it would be difficult to determine

whether or not there were any differences in kinetics. It can be seen that the activity of the

external NADH dehydrogenase in the presence of CCCP (2 M) is the same as the activity

of the external NADH dehydrogenase in the absence of uncoupler as the data points of both

energetic states overlap. Contrary to what was suggested previously [93] the SDH reducing

pathway kinetics are dependent on energy status of the IMM. The external NADH

dehydrogenase reducing pathway kinetics did not show any such dependency.

Figure 4.15 Combined state 2 and uncoupled reducing pathway kinetics with NADH as a substrate, Qr/Qt vs.

vO2. Reducing pathway kinetics under state 2 () and uncoupled conditions () were determined by titrating

with antimycin A (0 – 40 nM), Qr/Qt and vO2 were measured simultaneously. The Q-pool was partially

reduced with sub-saturating amounts of NADH (~14 M) using the NADH regenerating method. Under

uncoupled conditions CCCP was pre-incubated at a concentration of 2 M. Mitochondrial protein used per

experiment was 0.5-0.7 mg.

137

4.2.4 Are the biphasic patterns due to cytochrome bc1 complex kinetics?

Upon comparison of the cytochrome pathway kinetics obtained with either NADH or

succinate as a substrate (see figures 4.9-4.12) it can be concluded that there are no substrate

dependent differences in kinetics. An implication of this is that the biphasic patterns seen

under state 3 (figures 4.6, 4.7 and 4.11) and under uncoupled conditions (figures 4.8 and

4.12) are not due to the activities of the dehydrogenases; i.e. it does not matter in which

way the Q-pool is being reduced. Hence the underlying cause for the biphasic oxidising

pathway kinetics must be somewhere downstream of the Q-pool. Since S. pombe

mitochondria do not express an alternative oxidase (see Figure 1.14) these kinetics must

then be a characteristic of cytochrome pathway activity. The first acceptor of reducing

equivalents from the Q-pool is the cytochrome bc1 complex (see section 1.2.6) which is

known to exist as a homodimer [235]. Interestingly enough there is no need for the

cytochrome bc1 complex to be dimeric in order to accommodate for the protonmotive Q-

cycle [1] as either monomer on its own can achieve this. Based on work done on isolated

yeast (S. cerevisiae) cytochrome bc1 complexes Trumpower proposed a reaction

mechanism in which only half of the monomers are active [33]. He hypothesized that the

cytochrome bc1 complex could become fully active due to an effect of a change in

protonmotive force or due to a high concentration of ubiquinol [33, 236]. The biphasic

pattern observed in S. pombe cytochrome pathway kinetics could be a reflection of the

regulatory characteristics of the yeast cytochrome bc1 complex.

Activity of the cytochrome bc1 complex can be determined by following the

reduction of ferricyanide spectrophotometrically [79, 237]. In mitochondrial isolations

electrons can be transferred from cytochrome c to ferricyanide present in the reaction

medium [237]. In order to avoid electron transfer through complex IV the mitochondria are

incubated in the presence of KCN. Change in absorbance at 420 nm [237] was measured in

solutions incubated with KCN with either potato or S. pombe mitochondria. The

ferricyanide reduction rate was titrated with sub-saturating amounts of NADH using the

NADH regenerating system. The aim of the experiments was to determine whether or not a

biphasic pattern would be present in the relationship between substrate concentration and

ferricyanide reduction rate in S. pombe mitochondria. Potato mitochondria were used for

138

comparison. In this experimental system the only ‘electron sink’ available should be

ferricyanide. Addition of KCN (1 mM) effectively blocks electron transfer to cytochrome c

oxidase, this was verified using an oxygen electrode setup in a parallel experiment during

the same day. In the presence of antimycin A (1.7 M) the cytochrome bc1 complex is

completely inhibited, this was also verified. When the cytochrome bc1 complex is inhibited,

no electrons can be transferred to cytochrome c and therefore no ferricyanide reduction can

take place under these conditions. Control experiments were done to verify whether

cytochrome c was the only electron donor present that was able to reduce the added

ferricyanide. When ferricyanide was added to 1 ml of yeast reaction medium (see section

2.1.1.7) in the cuvette no change of absorbance at 420 nm was observed; the presence of

any of the following substances: KCN (1 mM), NADH (1.8 mM), Glucose-6-phosphate

dehydrogenase (10 units), glucose-6-phosphate (2.25 mM) and antimycin A (1.7 M) did

not lead to any change in absorbance (results not shown). Ferricyanide is not reduced by the

reaction medium or by any of the chemicals which are used during the experiments. Figure

4.16 shows a representative trace of an experiment using fresh potato mitochondria. With

just mitochondria and ferricyanide in the reaction medium no change in absorption is seen.

Upon addition of a saturating amount of NADH (1.8 mM) the reaction starts and within a

few seconds a steady state change in absorption can be seen. Upon addition of antimycin A

(60 ng) the reduction of ferricyanide was stopped completely. Similar results were obtained

with the sp.011 wt mitochondria (results not shown). From these experiments it was

concluded that in the presence of mitochondria and absence of substrate, there was no

ferricyanide reduction. Addition of antimycin A completely stopped the reduction of

ferricyanide, therefore the only pathway for electron transfer to ferricyanide is via

cytochrome c.

Figure 4.17 shows [NADH] vs. ferricyanide reduction rate in fresh potato

mitochondria under uncoupled conditions; the relationship displays a convex shape. Figure

4.18 shows [NADH] vs. ferricyanide reduction rate in sp.011 wt mitochondria under

uncoupled conditions, the relationship shows a biphasic pattern. These results suggest that

the biphasic patterns observed under conditions of a lowered or dissipated ∆ are likely to

be due to cytochrome bc1 complex kinetics.

139

Figure 4.16 Ferricyanide reduction measured spectrophotometrically in potato mitochondria.

Ferricyanide reduction was measured at 420 nm. The figure shows the change in absorption upon addition of

NADH (2mM) and antimycin A (60 ng), the cuvette was incubated with 1 ml potato reaction medium (see

section 2.1.3.2), 1 mM ferricyanide, 1 mM KCN and 0.2 mg of mitochondrial protein. Ferricyanide reduction

rate increases upon addition of NADH and addition of AA completely stops ferricyanide reduction, indicating

that there are no other pathways for electrons present but the cytochrome pathway. In order to add NADH and

AA the cover of the spectrophotometer had to be opened upon which recording stops temporarily until the

cover is closed again.

The results in figures 4.17 and 4.18 demonstrate a clear difference in kinetics between

potato and sp.011 wt mitochondria. Furthermore the sp.011 wt data suggests that upon

increasing concentrations of NADH (which reflects an increase in Qr/Qt) a biphasic pattern

occurs. These results corroborate the hypothesis that the biphasic pattern seen in sp.011 wt

respiratory kinetics are possibly due to bc1 complex activity. Equally also, it demonstrates

the presence of a biphasic pattern in the respiratory kinetics using a technique different

from the Q-electrode and the TPP+-electrode. The results shown in figures 4.17 and 4.18

show the relationship between NADH concentration and ferricyanide reduction rate.

Increasing the concentration of NADH leads to an increase in Qr/Qt (cf. figures 4.4, 4.6 and

4.8). Originally, duplicate experiments were planned where in the presence of KCN and

ferricyanide the relationship between NADH concentration and Qr/Qt could be determined.

Unfortunately both KCN and ferricyanide react strongly with the Q-electrode and these

compounds cannot be used in combination with this electrode. It is therefore not possible to

establish quantitatively the Q-redox poise in the spectroscopy experiments.

1.1

1.15

1.2

1.25

1.3

1.35

1.4

1.45

1.5

0 20 40 60 80 100 120 140

A42

0

time (s)

NADH

AA

140

Figure 4.17 Ferricyanide reduction rates under state 2 and uncoupled conditions in potato mitochondria.

Under both conditions the cuvette was incubated with 1 ml potato reaction medium, 1 mM KCN, 1 mM

ferricyanide, 1 mM G6P and 10 units of G6PD, under uncoupled conditions 2 M CCCP was present. A

steady state ferricyanide rate was induced by addition of a sub-saturating amount of NADH (0 – 100 M).

One rate was determined per experiment. The data points are averaged rates from 2 mitochondrial isolations.

Amount of mitochondrial protein used per experiment 0.1-02 mg.

Figure 4.18 Ferricyanide reduction rates under uncoupled conditions in sp.011 wt mitochondria. the cuvette

was incubated with 1 ml yeast reaction medium, 1 mM KCN, 1 mM Ferricyanide, 1 mM G6P, 10 units of

G6PD and 2 M CCCP. A steady state ferricyanide rate was induced by addition of a subsaturating amount of

NADH. One steady state rate was determined per experiment. The data points are averaged rates from 3

mitochondrial isolations. Amount of mitochondrial protein used per experiment 0.1 - 015 mg.

0

100

200

300

400

500

0 100 1 10-4 2 10-4 3 10-4 4 10-4 5 10-4 6 10-4

ferr

icy

an

ide

red

uct

ion

ra

te (

nm

ol

/ m

in /

mg

pro

tein

)

[NADH] M

0

100

200

300

400

500

600

700

0 100 5 10-5 1 10-4 1.5 10-4 2 10-4 2.5 10-4 3 10-4

ferr

icy

an

ide

re

du

cti

on

ra

te(n

mo

l / m

in /

mg

pro

tein

)

[NADH] M

141

The biphasic pattern in S. pombe mitochondrial respiratory kinetics may be explained in

terms of yeast bc1 complex kinetics as reported by Trumpower et al. [33, 236]. The results

obtained by Trumpower et al. were obtained from purified bc1 complexes isolated from S.

cerevisiae mitochondria. If indeed the biphasic patterns are caused by yeast bc1 kinetics

then it should be possible to reproduce the S. pombe findings in S. cerevisiae mitochondria.

4.2.5 Are biphasic respiratory kinetics a characteristic of yeast

mitochondria?

Figure 4.19 shows cytochrome pathway kinetics in mitochondria isolated from S. cerevisiae

mitochondria. Oxygen consumption rate and Qr/Qt were measured simultaneously. Under

state 2 conditions the relationship was linear (cf. Figure 4.4) whereas under uncoupled

conditions a biphasic pattern is present (cf. Figure 4.8). These results are comparable to

what was found in sp.011 wt mitochondria, which suggests that the biphasic respiratory

kinetics are indeed a characteristic of yeast mitochondria.

Figure 4.19 State 2 and uncoupled cytochrome pathway kinetics in S. cerevisiae mitochondria.

State 2 () and uncoupled () cytochrome pathway kinetics were determined using the NADH regenerating

system, titration range of NADH 0 – 55 M. Under uncoupled conditions CCCP was incubated at a

concentration of 2 M. Amount of mitochondrial protein used 1.2 mg. The state 2 and uncoupled data were

taken from 2 and 2 traces respectively, all data points are from one preparation.

0

10

20

30

40

50

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

rote

in

Qr/Qt

142

4.3 Discussion

4.3.1 Respiratory characteristics of S. pombe mitochondria—In this study mitochondria

isolated from S. pombe cells were characterised and the mitochondrial membrane potential

was determined for the first time. Upon addition of NADH or succinate (with ATP and

glutamate incubated) a ∆ is generated of about -200 mV (figures 4.1 and 4.2), addition of

a limited amount of ADP leads to a state 3 state 4 transition and addition of CCCP leads to

an increase in vO2 and dissipation of ∆. Additions of oligomycin or CAT prevent

generation of a state 3 upon subsequent addition of ADP. These responses are essentially

the same as is seen in plant [157] and mammalian [1] mitochondria. RCR values obtained

with NADH as a substrate are different from the values obtained with succinate as a

substrate. This is due to substrate dependent differences in reducing pathway kinetics (see

section 4.2.3); i.e. respiratory rates under state 3 and uncoupled conditions with NADH as a

substrate are considerably higher than with succinate as a substrate whereas the respiratory

rates under ADP limited conditions with either succinate or NADH as a substrate are

approximately the same. Upon addition of ADP or CCCP is decreased and cytochrome

pathway activity is stimulated. This is reflected in an approximately threefold increase in

rate under state 3 conditions and an almost six times increase under uncoupled conditions

with NADH as a substrate. A decrease in not only stimulates the cytochrome pathway,

it also deactivates SDH, whereas it has no effect on external NADH dehydrogenase activity

(figures 4.13 and 4.15), hence the low RCR values obtained with succinate.

Since S. pombe mitochondria do not express complex I there are only two coupling

sites available to generate a pmf, which are both in the cytochrome pathway (complex III

and IV) downstream from the Q-pool. Since SDH and the external NADH dehydrogenase

feed electrons directly into the Q-pool a maximum ADP/O value of 1.5 would be expected

[1]. The measured ADP/O values (NADH: 1.36, succinate: 1.22) are somewhat lower. A

lowered ADP/O value can have a wide variety of causes such as transport of inorganic

cations and various anionic metabolites, energy dissipation through uncoupling proteins,

proton pump ‘slips’ which would alter the H+/O ratio and the endogenous ion leak [238].

It could be that the current method of isolation yields mitochondria which have an IMM

which is relatively permeable for protons. Alternatively it could be that a low ADP/O value

143

is just a reflection of S. pombe physiology where mitochondria are not the primary source

for ATP generation. This however would be in contrast with reported ADP/O values for S.

pombe mitochondria by Jault et al. [105] (1.55-1.95) and Moore et al. [104] (~1.8) with

NADH as a substrate. The main difference between the mitochondrial isolation methods

used by those authors and the one used in this study lies within the enzyme digestion step

(see section 2.1.1.5). Most yeast cell walls contain -linked glucan, which can be digested

by gastric juice from the snail Helix pomatia [105]. S. pombe cell walls however also

contain -linked glucan [239] which is resistant to -glucanases from snail gastric juice.

Moore and Jault used the now no longer commercially available Novozyme 234 which has

-glucanase activity. For this study a combination of enzymes was used: zymolyase 20/T

which is only partially effective in producing spheroplasts in combination with lysing

enzymes [26]. Incubation at 30 C with both enzyme mixtures present leads to 90%

spheroplast production. It could be that the quality of the IMM also is affected by this

treatment to a greater extent than with Novozyme 234. At some stage zymolyase became

temporarily unavailable. As an alternative digestive enzyme Lyticase (Sigma) [240] was

investigated. Unfortunately in order to be effective, very long incubation steps (several

hours) and large quantities of the enzyme were needed, which made Lyticase unsuitable for

the purpose of digesting the cell wall in S. pombe mitochondria. No other enzymes were

investigated in this study. The hypothesis that digestion with zymolyase 20/T and lysing

enzymes leads to decreased IMM quality provides the basis for future work in which the

effectiveness of alternative enzymes can be investigated. Hopefully in the future a

commercial product will become available which is as effective as Novozyme 234.

Addition of either antimycin A, myxothiazol (both acting on complex III [1]) or

KCN (acting on complex IV [1]) can completely inhibit respiration. This indicates that

AOX is not expressed in S. pombe cells. This is a necessary requirement for these

mitochondria to be used as a model system in which AOX is heterologously expressed. The

presence of a cyanide-insensitive pathway in S. pombe mitochondria has been reported

before but this has later been ascribed to a contamination of the isolated mitochondria

[104], other studies on S. pombe mitochondria [26, 104, 105] did not show any cyanide-

insensitive respiration.

144

Membrane potential values obtained with either NADH or succinate20

were approximately

the same (see Table 4.1E). The decrease in upon addition of ADP was 20 mV which is

comparable with mitochondria from other sources [157]. Addition of nigericin did not lead

to a detectable change in TPP+ signal, this suggests that the pmf in S. pombe mitochondria

essentially consists of a , which is, as in plant mitochondria [157], probably due to the

presence of a K+/H

+ exchanger. It was postulated by Peter Mitchell that antiporters should

be present in the IMM which electroneutrally exchange cations for protons in order to

prevent osmotic swelling; the existence of the K+/H

+ exchanger

21 was demonstrated in

Keith Garlid’s laboratory [188]. At present there are no studies which show that S. pombe

mitochondria express a K+/H

+ exchanger in the IMM. However, the presence of this

exchanger in the IMM of S. cerevisiae mitochondria has been demonstrated [241] and its

gene sequence is known (YOL027c). A BLAST search in which the YOL027c gene

sequence was compared to the S. pombe genome revealed a homolog (SPAC23C11.17)

which is annotated on http://www.genedb.org/ as a mitochondrial inner membrane protein

involved in potassium ion transport. Its role was inferred from homology with YOL027c.

This indicates, in all likelihood, that S. pombe expresses a K+/H

+ exchanger in the IMM of

its mitochondria. These findings form the basis for future work. In a recent study by

Froschauer et al. [241] the existence of the K+/H

+ exchanger in S. cerevisiae was

demonstrated in an elegant set of experiments using submitochondrial particles (SMP’s)

obtained from S. cerevisiae mitochondria. The SMP’s were loaded with either K+-sensitive

or H+-sensitive fluorescent dyes. For determination of an anionic dye which readily

permeates depolarised membranes was used. K+ and H

+ translocations across the membrane

were inferred from changes in fluorescence. The experiments showed that K+ and H

+

movement across the membrane as a reaction to imposed concentration gradients of either

pH or K+ only occurred when K

+ was exchanged for H

+, transport of either species did not

occur independently. No change in membrane potential was detected during exchange of

K+ for H

+ demonstrating the electroneutrality of this process. Electrochemical equilibration

between [K+]

and [H

+]

brought about by nigericin was nearly identical to the equilibration

20

SDH fully activated with ATP and glutamate. 21

This exchanger is rather unselective and will in fact transport all alkali cations at similar rates, it is

referred to as the K+/H

+ exchanger to distinguish it from the Na

+/H

+ exchanger which is selective for

Na+ [188].


145

achieved by the endogenous system. Furthermore the process of K+/H

+ exchange was

sensitive to either quinine, DCCD or Mg2+

, that are known inhibitors of this process. A

mutant strain which lacks the gene for the K+/H

+ exchanger (yol027) was not able to

exchange K+ for H

+. Expression of the human gene LETM1 (which is a homolog of

YOL027c) restored the K+/H

+ exchange [241]. The experiments described above could

readily be performed on S. pombe derived SMP’s. If, as expected, the presence of a K+/H

+

exchanger in S. pombe mitochondria can be demonstrated then subsequently S. pombe

mitochondria could be used as a model system in medical research. It has been reported that

a hemizygous deletion of the LETM1 gene in humans causes Wolf-Hirschhorn disease

[241].

It is apparent from figures 4.1 and 4.2 that upon multiple additions of small aliquots

of ADP successive state 4 values are lowered whereas the corresponding vO2 values

are increased. This behaviour is reminiscent of potato mitochondria in which two molecules

of ADP are generated (from ATP and AMP) via adenylate kinase activity in the

intermembrane space [110]. It has been shown that under ADP limited conditions there is a

continuous synthesis of ATP in liver and potato mitochondria [110, 186]. Addition of

inhibitors of the ATP synthase or adenylate kinase under ADP limited conditions led to an

increase in and concomitant decrease in vO2 [111]. With each addition of ADP the total

concentration of adenine nucleotides in the medium increases, therefore with each

successive state 4 there is more ATP available for ADP generation via adenylate kinase.

Since the presence of adenylate kinase has been shown in S. pombe mitochondria [112] it

was an attractive notion to link the observed increased IMM conduction under state 4

conditions in S. pombe mitochondria to adenylate kinase activity. However, adenylate

kinase activity could not be demonstrated conclusively. Under state 4 conditions the

effect(s) of adding the following substances were studied: AMP, oligomycin, CAT and

Ap5A (data not shown). Addition of oligomycin led to a decrease in vO2, however no

change in was seen. Addition of Ap5A (an inhibitor of the adenylate kinase [21]) did

not appear to have any effect on either or vO2. Addition of CAT led to a decrease in

vO2 but no effect on was seen. Addition of AMP had no effect. The decrease in vO2

under state 4 conditions observed when either oligomycin or CAT was added indicates that

ATP was being synthesized at that time, this however should have been reflected also in a

146

slight increase in . In potato mitochondria addition of AMP leads to the generation of a

state 3 because ADP was generated via the adenylate kinase using AMP and ATP as

substrates [21]. This was not seen in S. pombe. Addition of Ap5A had no effect. From these

results it is unclear if adenylate kinase is active in S. pombe mitochondria. A BLAST search

through the S. pombe genome (http://www.genedb.org/) does not reveal a mitochondrial

adenylate kinase. Upon scrutinizing [112] it becomes apparent that no experimental

evidence is given to support the original claim that S. pombe expresses a mitochondrial

adenylate kinase. Based on experimental evidence obtained during this thesis work it is the

authors opinion that S. pombe mitochondria do not express AK. What causes the increase

in respiratory rates under state 4 conditions in S. pombe mitochondria remains unclear at

present.

4.3.2 Are the biphasic patterns due to an experimental artefact?—The biphasic cytochrome

pathway kinetics seen under state 3 conditions, Qr/Qt vs. vO2 and ∆ vs. vO2, (figures 4.6

and 4.7) and under uncoupled conditions, Qr/Qt vs. vO2, (Figure 4.8) have not been

observed previously in mitochondria. It has been reported that in cyanide-sensitive

mitochondria, electron flow through the cytochrome pathway is linearly dependent upon

the redox state of the Q-pool [234]. To rule out whether or not the biphasic pattern is an

artefact all possible non-physiological causes were investigated. When growing yeast

cultures there is always the possibility of contamination (bacterial or the presence of

another yeast). If a contamination was detected (cells being round instead of rod-like for

instance) the yeast culture was discarded and all solutions and yeast plates were replaced.

Yeast culture contamination occurred only rarely (two or three times during this study). To

exclude the possibility that the biphasic respiratory kinetics were due to unnoticed22

contamination of the S. pombe cultures determination of cytochrome pathway kinetics was

repeated many times. State 3 , Qr/Qt vs. vO2 data are from eleven traces from five different

mitochondrial isolations. State 3, ∆ vs. vO2 data are from five traces from three

mitochondrial isolations and the uncoupled, Qr/Qt vs. vO2 data are from five traces from

five mitochondrial isolations. The mitochondrial preparations were isolated from yeast

22

Highly unlikely as the cultures were discarded if there was any doubt. Also, Dr M. Albury was

always very helpful in giving a second opinion.


147

cultures grown from two different yeast plates. A biphasic pattern is seen under state 3 or

uncoupled conditions, but not under state 2 or state 4 conditions, this indicates that the

cytochrome pathway kinetics show a dependency on energy status which is reflected in the

biphasic pattern. If these biphasic kinetics were due to an experimental artefact why do the

biphasic kinetics not appear under all energetic conditions? The biphasic pattern is seen in

the relationships between Qr/Qt vs. vO2(state 3 and uncoupled) and vs. vO2 (state 3),

which means that the same pattern is seen in data sets from measurements done with

different electrodes. The biphasic pattern cannot be attributed to the use of the same

reference electrode since different ones were used in the Q-electrode set-up and the TPP+

electrode set-up. The biphasic pattern in S. pombe is also seen with succinate as a substrate

thereby excluding the possibility that the biphasic pattern is due to the NADH titration

method. The biphasic pattern is not seen in potato or arum mitochondria (see chapter 5)

using the exact same experimental set-up which was used with the S. pombe system. A

biphasic pattern was also seen using a completely different technique (spectrophotometry).

In mitochondria expressing AOX the oxidising pathway kinetics under state 3 and

uncoupled conditions were different (to be discussed in chapter 5). Addition of an extra

oxidase led to a change in the biphasic pattern which indicates that it is a physiological

process rather than an experimental artefact. In conclusion, the biphasic patterns as seen

under state 3 and uncoupled conditions in the cytochrome pathway kinetics are a

characteristic of S. pombe mitochondria.

4.3.3 Are the biphasic patterns due to cytochrome bc1 complex kinetics?—It was

determined that the oxidising pathway kinetics in S. pombe mitochondria are the same with

either NADH or succinate as a substrate (up until the point where vO2 rates become

limiting with succinate dependent respiration); see figures 4.9 – 4.12. In other words, it

does not matter in which way the Q-pool is reduced. That means that the underlying cause

of the biphasic patterns in the oxidising pathway kinetics must be located downstream of

the Q-pool. Since S. pombe mitochondria do not express AOX the biphasic kinetics must be

a characteristic of the cytochrome pathway. The cytochrome pathway consists of the

cytochrome bc1 complex, cytochrome c and cytochrome c oxidase with a stoichiometry of 3

: 14 : 7 [7]. On the basis of contemporary work done by Trumpower et al. on isolated yeast

148

cytochrome bc1 complexes (see section 4.2.4) it was decided to investigate the possible role

of the cytochrome bc1 complex in S. pombe mitochondria as a cause for the biphasic

cytochrome pathway kinetics. For this purpose a different experimental approach was used.

As opposed to simultaneously determine vO2 and either Qr/Qt or (using the oxygen

electrode in combination with either the Q-electrode or the TPP+-electrode) a

spectrophotometic technique was used. Ferricyanide (Fe(CN)63-

) is an artificial electron

acceptor which can be reduced by cytochrome c. Mitochondria were incubated with

ferricyanide, KCN (to inhibit cytochrome c oxidase), G-6-P and G-6-P-dehydrogenase

(NADH regenerating system). It was determined that ferricyanide was the only pathway

available to electrons to leave the respiratory chain (see Figure 4.16). Upon addition of

subsaturating amounts of NADH the steady state change in absorption at 420 nm was

determined. Increasing concentrations of NADH led to an increase in absorption change

which reflects increased reduction of ferricyanide. This was done for both potato and S.

pombe mitochondria under uncoupled conditions, see figures 4.17 and 4.18. It is apparent

that the relationship of [NADH] vs. ferricyanide reduction rate in potato mitochondria has a

convex shape whereas in S. pombe mitochondria there appears to be a biphasic pattern.

These results indicate that, again, under uncoupled conditions there are kinetic differences

in cytochrome pathway kinetics between potato and S. pombe mitochondria. Furthermore,

the shape of the S. pombe kinetics appears to be biphasic, at low [NADH] there is low

activity until a threshold value is reached upon which activity increases disproportionally.

From the data available, this threshold NADH concentration value cannot be gauged, more

experiments are needed in order to get a more continuous set of data points. It was

concluded that it is probably cytochrome bc1 complex activity which causes the non-linear

state 3 and uncoupled cytochrome pathway kinetics in S. pombe mitochondria.

4.3.4 Are biphasic respiratory kinetics a characteristic of yeast mitochondria?—The

hypothesis that the biphasic respiratory kinetics in S. pombe mitochondria are due to bc1

complex kinetics was based on research done by Trumpower et al. on isolated bc1

complexes from S. cerevisiae mitochondria [33, 236]. If this hypothesis is correct then the

biphasic respiratory kinetics should also be present in S. cerevisiae mitochondria. It was

shown that under uncoupled conditions indeed the cytochrome pathway kinetics in S.

149

cerevisiae mitochondria display a biphasic pattern, whereas under ADP limited conditions

the relationship between Qr/Qt and vO2 is linear. These findings agree perfectly with the

results obtained from S. pombe mitochondria and they corroborate the hypothesis that the

observed biphasic patterns are caused by the bc1 complex. In order to understand how the

kinetics of a single protein complex might determine mitochondrial respiratory kinetics

Trumpower’s work on the S. cerevisiae bc1 complex will be reviewed in the general

discussion chapter. Mechanisms proposed by Trumpower will be related to the biphasic

respiratory kinetics found in S. pombe mitochondria.

Having thoroughly characterised the respiratory kinetics of the S. pombe wild type

mitochondria the next step was to incorporate the alternative oxidase protein in order to

investigate its influence on respiratory kinetics.

150

Chapter 5

Functional expression of the alternative oxidase in


5.1 INTRODUCTION

Schizosaccharomyces pombe mitochondria heterologously expressing the alternative

oxidase from the plant Sauromatum guttatum have been used in this laboratory to

investigate structure-function relationships [59, 63, 94]. Many plant species as well as

certain fungi23

and protists display cyanide-insensitive respiration [66]. The electron

transfer chain in mitochondria of these species is branched at the level of the Q-pool, see

Figure 1.4. Two pathways can accept reducing equivalents from the Q-pool; the

conventional protonmotive cytochrome pathway and the non-protonmotive alternative

pathway, which consists of the single AOX protein, see section 1.2.11. When the

cytochrome pathway is inhibited (e.g. through incubation with antimycin A) several AOX

expressing organisms can still generate a p through complex I [66]. S. pombe

mitochondria however do not express complex I (see Figure 1.12) therefore S. pombe

mitochondria expressing AOX, in the presence of an inhibitor of the cytochrome pathway

cannot generate a p. It was found in plant mitochondria expressing AOX, when incubated

in the presence of antimycin A, that succinate dependent respiration does not lead to the

generation of p [176]. It was traditionally assumed that the alternative pathway

functioned as an overflow pathway for reducing equivalents under conditions where

cytochrome pathway activity was limited [37]. It has been shown however that both

pathways can compete with each other for reducing equivalents [79]. Reducing equivalents

can be diverted from the cytochrome pathway to the alternative pathway and vice versa

[237]. Earlier work in this laboratory showed that under state 3 and state 4 conditions AOX

23

But not S. cerevisiae [242] or S. pombe [104].

151

expression in S. pombe mitochondria led to a decrease in cytochrome pathway activity. In

that study [93] oxygen consumption rate and Qr/Qt were measured simultaneously,

however was not determined. As shown in chapter 4, S. pombe mitochondria, respiring on

either succinate or NADH can generate a (see figures 4.1 and 4.2). The membrane

potential in these mitochondria shows typical responses upon addition of ADP or uncoupler

as seen in mitochondria from other yeasts [160] and from other species [1, 157]. It was

concluded by Affourtit et al. that expression of AOX in S. pombe mitochondria leads to

competition between the alternative and the cytochrome pathway for reducing equivalents,

which leads to a lowered efficiency of energy transduction [93]. The same study showed a

phenotypic effect of AOX expression in S. pombe, both growth rate and yield of S. pombe

batch cultures were lowered when grown in the absence of thiamine (AOX expressed, see

section 2.1.1). It was suggested that the change in phenotype was due to the effect of AOX

expression on S. pombe respiratory kinetics [93]. It has been determined previously in this

laboratory that S. guttatum AOX expressed in S. pombe mitochondria cannot be stimulated

by pyruvate [59]. More recently it was found that S. guttatum AOX expressed in S. pombe

mitochondria is constitutively active, as opposed to A. thaliana AOX expressed in S. pombe

mitochondria, which can be stimulated by pyruvate [70]. It was hypothesized that during

development of thermogenic species (like S. guttatum) high AOX engagement is ensured,

at least in part, by expression of constitutively active isozymes [70]. In this chapter the

respiratory kinetics of five types of mitochondria have been investigated, see Table 5.1.

type source

sp.011 wt non-transformed S. pombe ALU

sp.011 AOX transformed S. pombe AOX expressed AU

sp.011 AOX + T transformed S. pombe AOX repressed AUT

sp.011 pREP transformed S. pombe empty vector AU

A. maculatum isolated from fresh and cold stored spadices

Table 5.1 types of mitochondria used and source material. A: adenine U: uracil L: leucine T: thiamine.

152

Mitochondria isolated from S. pombe cells grown in the presence of adenine, leucine and

uracil are referred to as sp.011 wt (wild type), see chapter 4. Mitochondria isolated from S.

pombe cells which have been transformed with a plasmid coding for the S. guttatum AOX

protein, grown in the presence of adenine and uracil, are referred to as sp.011 AOX.

Mitochondria isolated from S. pombe cells which have been transformed with a plasmid

coding for the S. guttatum AOX protein, grown in the presence of adenine, uracil and

thiamine are referred to as sp.011 AOX + T (thiamine); AOX expression is repressed in

these mitochondria due to the presence of the nmt1 promoter on the plasmid. Mitochondria

isolated from S. pombe cells which have been transformed with the pREP plasmid (a vector

lacking the gene coding for AOX), grown in the presence of adenine and uracil are referred

to as sp.011 pREP.

In previous work done in this laboratory the effects of a functionally expressed

alternative oxidase in S. pombe mitochondria on respiratory kinetics were investigated [93].

Oxidising pathway kinetics were determined by titrating succinate dependent respiration

with malonate under various energetic conditions. Limited data obtained with NADH as a

substrate was found to overlap with the succinate data and it was concluded that there are

no substrate dependent differences in oxidising pathway kinetics in S. pombe mitochondria

expressing AOX. The amount of data obtained in that study was limited and the

mitochondria used were of a lesser quality than the ones used in this study (see section

5.2.1). In this study the oxidising pathway kinetics were determined with NADH as a

substrate using the NADH regenerating system (see section 2.4.4.4) in S. pombe

mitochondria of good quality (see section 4.2.1 and 5.2.1). The oxidising pathway kinetics

obtained with NADH as a substrate were different from the kinetics obtained by Affourtit et

al. [93]. To investigate possible substrate dependent differences, oxidising pathway kinetics

with succinate as a substrate were determined. Possible cause(s) for the differences between

the results of this study and those of Affourtit were investigated.

It was found that efficiency of energy transduction and cytochrome pathway activity

was lowered in S. pombe mitochondria expressing AOX [93]. To investigate a possible

decrease in protonmotive force due to AOX activity, was determined in S. pombe

mitochondria expressing AOX. For comparison with a system in which AOX is naturally

expressed the respiratory kinetics of mitochondria isolated from A. maculatum were

153

investigated. To investigate the effects of AOX expression on S. pombe respiratory kinetics

results from experiments done on sp.011 AOX mitochondria (expressing AOX) were

compared to results from the control group of sp.011 AOX+T mitochondria (AOX

repressed). The act of transformation, possibly, could have altered respiratory kinetics of S.

pombe mitochondria; this was investigated by comparing the results obtained with sp.011

AOX and AOX+T mitochondria to results obtained with sp.011 wt (the wild type, see

chapter 4) mitochondria and with sp.011 pREP mitochondria (transformed S. pombe

mitochondria with a vector lacking the AOX gene).

5.2 RESULTS

5.2.1 General characterisation of sp.011 AOX, AOX + T, pREP and wt respiratory

kinetics Tables 5.2 and 5.3 show oxygen consumption rates and RCR values for sp.011

AOX, AOX + T, pREP and wt mitochondria. It was a priori expected that the respiratory

characteristics of the sp.011 wt, sp.011 pREP and sp.011 AOX + T mitochondria would be

similar, given that these mitochondria only have one oxidising pathway, the cytochrome

pathway, as opposed to sp.011 AOX mitochondria which have both a cytochrome and an

alternative pathway. sp.011 wt and pREP mitochondria have comparable oxygen

consumption rates and RCR values. The sp.011 AOX + T oxygen consumption rates and

RCR values are however different from the sp.011 wt and pREP values. Under ADP

limited conditions the oxygen consumption rates of sp.011 wt, pREP and AOX+T are

comparable. But under conditions where the protonmotive force is decreased (under state 3

and uncoupled conditions) the sp.011 AOX+T oxygen consumption rates are considerably

lower than those of sp.011 wt and pREP. Also the sp.011 WT and pREP mitochondria

appear to be better coupled than the sp.011 AOX+T mitochondria based on the RCR

values. In order to compare sp.011 AOX cytochrome pathway activity with the other three

types, oxygen consumption rates and RCR values were determined in the presence of octyl

gallate (which potently inhibits AOX [59]). The obtained vO2 and RCR values are

comparable to the sp.011 AOX+T values but are lower than the sp.011 wt and pREP

values.

154

sp.011 AOX Respiratory Rate (nmol O2 min-1

mg-1

protein)


NADH 199 (27) 328 (34) 224 (13) 531 (82)

NADH + OG 92 (10) - - 396 (43)

NADH + AA 81 (17) - - -

succinate 142 (14) 198 (13) 136 (8) 176 (15)

succinate + OG 83 (12) 123 (21) 72 (6) -

succinate + AA 84 (13) - - -

sp.011 AOX + T

NADH 80 (13) 169 (29) 72 (9) 398 (55)

NADH + OG 77 (12) - - 350 (48)

succinate 83 (10) 136 (16) 87 (10) 124 (20)

sp.011 pREP

NADH 96 (13) 245 (25) - 543 (55)

succinate 100 (13) 196 (24) 107 (23) -

sp.011 wt

NADH 91 (13) 281 (39) 110 (22) 537 (68)

succinate 106 (15) 156 (18) 97 (7) 144 (25)

Table 5.2 Respiratory rates in sp.011 AOX, AOX + T, wt and pREP mitochondria under various energetic

conditions and various substrates. Format: rate ( S.D.). See appendix 2B for full details on amount of

mitochondrial isolations and experimental traces. Concentrations of chemicals added: NADH (1.8 mM),

succinate (9 mM), octyl gallate (14 M), antimycin A (~40 nM). Uncoupled conditions are defined as

respiratory activity in the presence of an uncoupler (2 µM CCCP in all cases), which dissipates the

protonmotive force leading to maximal activation of the cytochrome pathway. The concentration of added

ADP to induce a state 3 to state 4 transition was 0.2 mM in all cases. State 3 conditions were induced by pre-

incubation of ADP (1 mM). All succinate rates are from mitochondria incubated in the presence of ATP (0.2

mM) and glutamate (9 mM). The energy status had no effect on AOX activity, therefore the sp.011 AOX rates

when incubated in the presence of antimycin A are the same under all energetic conditions.

155

Based on similarities in vO2 rates and RCR values the various S. pombe mitochondria are

divided into two groups. sp.011 wt and pREP mitochondria are well coupled (with NADH

as a substrate the uncoupled / state 2 RCR is ~6) and display high uncoupled NADH vO2

rates ~ 540 nmol O2 / min / mg protein. sp.011 AOX (incubated in the presence of octyl

gallate) and AOX+T mitochondria have somewhat lower couplings (with NADH as a

substrate the uncoupled / state 2 RCR is 4-5) and uncoupled NADH vO2 rates are in the

range of 350-400 nmol O2 / min / mg protein. sp.011 AOX mitochondria in the absence of

octyl gallate (both pathways active) display higher vO2 rates under all energetic conditions

when compared to sp.011 AOX+T mitochondria. Also the RCR with NADH as a substrate

(uncoupled / state 2) is considerably lower (2.7) when compared to sp.011 AOX+T (5)

indicating that AOX activity affects the efficiency of energy transduction in S. pombe

mitochondria.

Table 5.3 RCR values for sp.011 wt, pREP, AOX and AOX + T mitochondria with either NADH or succinate

as substrate. NADH (1.8 mM) succinate (9 mM). The RCR values (see section 2.4.4.3) in Table B were

calculated using the vO2 values from table 5.1. All succinate rates are from mitochondria incubated in the

presence of ATP (0.2 mM) and glutamate (9 mM).

sp.011 wt RCR's

substrate S3 / S4 uncoupled / S2

NADH 2.6 5.9

succinate

1.6 1.4

sp.011 pREP

NADH - 5.7

sp.011 AOX

NADH 1.5 2.7

NADH + OG - 4.3

succinate

1.5 1.2

sp.011 AOX + T

NADH 2.3 5

NADH + OG - 4.5

succinate

1.6 1.5

156

It is not readily apparent why the cytochrome pathway respiratory characteristics of sp.011

AOX (incubated in the presence of OG) and sp.011 AOX+T mitochondria are different

from sp.011 wt and sp.011 pREP cytochrome pathway kinetics. The act of transformation

itself does not affect rates or coupling since the values obtained with sp.011 pREP compare

well with sp.011 wt. The presence of thiamine in the yeast growth medium cannot be the

cause for lowered respiratory rates and lowered coupling seen in sp.011 AOX+T (compared

to sp.011 wt) since sp.011 AOX mitochondria (incubated in the presence of OG) show rates

and couplings comparable to sp.011 AOX+T. The presence of leucine (in sp.011 wt) cannot

be the causal factor for higher respiratory rates and coupling, compared to sp.011 AOX+T

and sp.011 AOX (incubated in the presence of OG) mitochondria, since sp.011 pREP rates

and coupling are comparable to sp.011 wt values. The only parameter which sp.011 AOX

and sp.011 AOX+T mitochondria have in common is the fact that both types of

mitochondria are isolated from S. pombe cells which have been transformed with a vector

coding for AOX. How this in itself could affect cytochrome pathway activity is unclear.

Importantly though, the cytochrome pathway characteristic of sp.011 AOX+T and sp.011

AOX mitochondria agree well with each other and the sp.011 AOX+T mitochondria

constitute a proper control group for comparison with results obtained from sp.011 AOX

mitochondria.

To verify that AOX indeed was expressed (or repressed) in the transformed

mitochondria, western blots were made (see section 2.2) using protein from isolated sp.011

AOX and AOX+T mitochondria. For comparison, protein from isolated A. maculatum

mitochondria was used, see Figure 5.1. The A. maculatum protein bands (gel A lanes 6 and

7, gel B lanes 5 and 7) display both oxidised (~75 kDa) and reduced (~32 kDa) forms of

AOX. It can be seen that AOX is expressed primarily in the reduced form (gel A, lanes 1-4

and gel B lanes 1-4) as indicated by the 32 kDa bands. The sp.011 AOX blots are marked

by several bands of lower molecular weight. This was seen before in prior research (cf.

Figure 5.1 in [26]) and it was attributed to protein degradation occurring during cold

storage (-20°C). In order to prevent this, mitochondrial protein samples were incubated

with a protease inhibitor cocktail (Roche Products Ltd) prior to cold storage, which clearly

did not work. Importantly though, the AOX antibodies used, bound to a ~32 kDa protein

band (which is the right size for an AOX monomer [243] ) and targeted both sp.011 AOX

157

and A. maculatum mitochondria, but not sp.011 AOX+T in which AOX expression is

repressed. Therefore sp.011 AOX mitochondria functionally express AOX.

A

B

Figure 5.1 Immuno-detection of AOX in S. pombe and A. maculatum mitochondria. Lanes were loaded with

15 µg of protein and probed with antibodies raised against AOX (see section 2.2). Gel A was loaded with:

sp.011 AOX mitochondria (1-4), sp.011 AOX + T mitochondria (5) and A. maculatum mitochondria (6-7).

Gel B was loaded with sp.011 AOX mitochondria (1-4) and A. maculatum mitochondria (5 and 7), lane 6 is

empty. Proteins were separated under non-reducing conditions.

158

0

20

40

60

80

100

120

140

0 0.2 0.4 0.6 0.8 1

vO2 n

mol

O2 /

min

/ m

g p

rote

in

Qr/Qt

5.2.2 Oxidising pathway kinetics with NADH as substrate

To obtain state 3 or uncoupled oxidising pathway kinetics a saturating amount of ADP (1

mM) or a saturating amount of CCCP (2 M) was incubated. To obtain state 4 oxidising

pathway kinetics a state 3 to state 4 transition was induced by adding an aliquot of ADP

(~50 M).

5.2.2.1 Comparing sp.011 pREP and wt cytochrome pathway kinetics with NADH as a

substrate—The act of transforming S. pombe cells itself could possibly lead to changes in

respiratory kinetics unrelated to the expression of AOX. To investigate an effect of

transforming S. pombe cells with a vector on mitochondrial respiratory kinetics,

cytochrome pathway kinetics of sp.011 wt and sp.011 pREP mitochondria were compared.

Figure 5.2 shows the relationship between Qr/Qt and vO2 under state 2 and state 4

conditions. The sp.011 pREP vO2 rates at every value of Qr/Qt are slightly higher than the

sp.011 wt rates. Both relationships are linear which agrees with results previously obtained

in this laboratory [104] with succinate as a substrate.

Figure 5.2 Combined state 2 and state 4 cytochrome pathway kinetics of sp.011 wt and sp.011 prep

mitochondria. Using the NADH regenerating system (titration range 0 – 75 M) Qr/Qt and vO2 were

measured simultaneously, sp.011 wt state 2 () and state 4 () data were taken from Figure 4.4. sp.011

pREP state 2 () data was obtained from three mitochondrial isolations, data points were combined from

three traces. Mitochondrial protein used per experiment is 0.5-0.7 mg.

159

0

50

100

150

100 120 140 160 180 200 220

vO

2 n

mo

l O

2 /

min

/ m

g p

ro

tein


Figure 5.3 shows the relationship between ∆ and vO2 under state 2 and state 4 conditions

in sp.011 pREP and sp.011 wt mitochondria respiring on NADH. The familiar non-ohmic

relationship is seen (see section 3.1.2). The data sets from sp.011 wt and sp.011 pREP

overlap, which suggests that the IMM conduction in both types of mitochondria is the same

and that the act of transformation did not have an effect on proton permeability of the IMM.

Figure 5.3 Combined state 2 and state 4 cytochrome pathway kinetics, ∆ vs. vO2, from sp.011 wt and

sp.011 pREP mitochondria. Using the NADH regenerating system (titration range 0 – 75 M) ∆ and vO2

were measured simultaneously. sp.011 wt state 2 () and state 4 () data were taken from Figure 4.5. sp.011

pREP state 2 () data were obtained from two mitochondrial isolations, combining data points from four

traces. Mitochondrial protein used per experiment is 0.5-0.7 mg.

Figure 5.4 shows the relationship between Qr/Qt and vO2 under state 3 conditions in sp.011

pREP and sp.011 wt mitochondria respiring on NADH. The sp.011 pREP vO2 rates at

every value of Qr/Qt are slightly higher than the sp.011 wt rates . The same biphasic pattern

as in sp.011 wt mitochondria can be seen.

160

0

50

100

150

200

250

300

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

ro

tein

Qr/Qt

Figure 5.4 Combined state 3 cytochrome pathway kinetics, Qr/Qt vs. vO2, from sp.011 wt and sp.011 pREP

mitochondria. Using the NADH regenerating system (titration range 0 – 75 M) Qr/Qt and vO2 were

measured simultaneously. sp.011 wt state 3 () data were taken from Figure 4.6. sp.011 pREP state 3 ()

data were obtained from three mitochondrial isolations, combining data points from five traces. Mitochondrial

protein used per experiment is 0.5-0.7 mg.


pREP and sp.011 wt mitochondria. The two data sets overlap but the typical biphasic

pattern, as seen in sp.011 wt mitochondria is not seen in this sp0.11 pREP data set. The

biphasic pattern was seen in sp.011 pREP data obtained with a TPP+-electrode that was not

properly calibrated, i.e. per tenfold change in [TPP+] ratio (see section 2.4.3) a value lower

than 59 was obtained. This only affects the magnitude of the values calculated and not

the shape of the relationship between and vO2 (see the inset in Figure 5.5). It can be

concluded that under state 3 conditions the relationship between and vO2 in sp.011

pREP is comparable to sp.011 wt.

161

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50

100

150

200

120 130 140 150 160 170 180

vO

2 n

mo

l O

2 /

min

/ m

g p

rote

in


0

20

40

60

80

100

120

155 160 165 170 175 180v

O2 n

mo

l O

2 /

min

/ m

g p

rote

in


Figure 5.5 Combined state 3 cytochrome pathway kinetics, ∆ vs. vO2, from sp.011 wt and sp.011 pREP

mitochondria. Using the NADH regenerating system (NADH titration range 0 – 75 M) Qr/Qt and vO2 were

measured simultaneously. sp.011 wt state 3 () data were taken from Figure 4.7. sp.011 pREP state 3 ()

data (ADP 1 mM) were obtained from one mitochondrial isolation, combining data points from two traces.

The inset shows the typical biphasic pattern which is also present in sp.011 pREP mitochondria, data from

one trace. Mitochondrial protein used per experiment is 0.5-0.7 mg.

Figure 5.6 shows the relationship between Qr/Qt and vO2 under uncoupled conditions in

sp.011 pREP and sp.011 wt mitochondria respiring on NADH. The sp.011 pREP vO2 rates

at every value of Qr/Qt are slightly higher than the sp.011 wt rates. The sp.011 pREP data

shows a biphasic pattern similar to the one seen in the sp.011 wt data. Under each energetic

condition in sp.011 pREP mitochondria the vO2 is a bit higher at each value of Qr/Qt. One

possible cause for this could be a difference in the quality of the IMM in both types of

mitochondria. However, similar RCR values and overlapping state 2 ∆ vs. vO2 data

suggest that both types of mitochondria have approximately the same IMM conductive

characteristics. With the exception of somewhat higher respiratory rates in sp.011 pREP

mitochondria it was concluded that the respiratory kinetics of sp0.11 wt and sp.011 pREP

mitochondria are essentially the same.

162

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100

200

300

400

500

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

ro

tein

Qr/Qt

Figure 5.6 Combined uncoupled cytochrome pathway kinetics, Qr/Qt vs. vO2, from sp.011 wt and sp.011

pREP mitochondria. Using the NADH regenerating system (NADH titration range 0 – 75 M) Qr/Qt and vO2

were measured simultaneously. sp.011 wt uncoupled () data were taken from Figure 4.8. sp.011 pREP state

3 () data (ADP 1 mM) were obtained from three mitochondrial isolations, combining data points from six

traces. Mitochondrial protein used per experiment is 0.5-0.7 mg.

It was concluded that transformation of S. pombe cells does not lead to any significant

changes in mitochondrial respiratory kinetics. It was already established that AOX is

correctly targeted to the IMM in S. pombe mitochondria expressing AOX [59]. Affourtit

found that the respiratory kinetics of sp.011 AOX mitochondria were different from those

of sp.011 AOX+T mitochondria, it was assumed that these differences were due to AOX

activity [93]. Having established that AOX is correctly targeted and that transformation

does not change the S. pombe wild type respiratory kinetics; in a next series of experiments

the oxidising pathway kinetics of sp.011 AOX and sp.011 AOX+T mitochondria respiring

on NADH were investigated.

163

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150

200

250

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

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g p

rote

in

Qr/Qt

5.2.2.2 Comparing sp.011 AOX and sp.011 AOX + T oxidising pathway kinetics with

NADH as a substrate—To investigate any effects of AOX expression on respiratory

kinetics, oxidising pathway kinetics of sp.011 AOX and sp.011 AOX+T mitochondria were

compared. Figure 5.7 shows the relationship between Qr/Qt and vO2 under state 2 and state

4 conditions in sp.011 AOX and sp.011 AOX + T mitochondria respiring on NADH. The

vO2 rates at any value of Qr/Qt in sp.011 AOX mitochondria are considerably higher than

in sp.011 AOX + T, i.e. the presence of AOX leads to an overall increase in vO2. Both

relationships are linear as seen previously in sp.011 wt and sp.011 pREP (cf. Figure 5.2)

Figure 5.7 Combined state 2 and state 4 oxidising pathway kinetics, Qr/Qt vs. vO2, from sp.011 AOX and

sp.011 AOX + T mitochondria. Using the NADH regenerating system Qr/Qt and vO2 were measured

simultaneously (NADH titration range 0 – 75 M). sp.011 AOX state 2 () data were obtained from five

mitochondrial isolations, combining data from eight traces. sp.011 AOX + T state 2 () and state 4 () data

were obtained from four and one mitochondrial isolations respectively, combining data points from eight and

two traces respectively. Mitochondrial protein used per experiment is 0.5-0.7 mg.

Figure 5.8 shows the relationship between Qr/Qt and vO2 under state 3 conditions in sp.011

AOX and sp.011 AOX+T mitochondria respiring on NADH. In sp.011 AOX mitochondria

the vO2 rates at each value of Qr/Qt are higher than in sp.011 AOX+T mitochondria.

Interestingly, in the presence of AOX the biphasic pattern is no longer seen and the

relationship between Qr/Qt and vO2 has become linear. The relationship between Qr/Qt and

164

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250

300

350

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

rote

in

Qr/Qt

vO2 in the sp.011 AOX + T mitochondria is biphasic as seen in sp.011 wt and sp.011 pREP

(cf. Figure 5.4).

Figure 5.8 Combined state 3 oxidising pathway kinetics, Qr/Qt vs. vO2, from sp.011 AOX and sp.011 AOX +

T mitochondria. Using the NADH regenerating system Qr/Qt and vO2 were measured simultaneously (NADH

titration range 0 – 75 M). sp.011 AOX state 3 () data were obtained from three mitochondrial isolations,

combining data from six traces. sp.011 AOX + T state 3 () data were obtained from seven mitochondrial

isolations, combining data points from twelve traces. Mitochondrial protein used per experiment is 0.5-0.7

mg.

Figure 5.9 shows the relationship between Qr/Qt and vO2 under uncoupled conditions in

sp.011 AOX and sp.011 AOX+T mitochondria respiring on NADH. In sp.011 AOX

mitochondria the vO2 rates at each value of Qr/Qt are higher than in sp.011 AOX+T

mitochondria. As seen under state 3 conditions (cf. Figure 5.8) the biphasic pattern appears

to have become linear in sp.011 AOX mitochondria. However, when looking at individual

experimental traces of sp.011 AOX mitochondria (see inset in Figure 5.9) it is evident that

a biphasic pattern is still to be seen, indicating that the activity of AOX leads to a masking

(but not disappearance) of the biphasic kinetics. The sp.011 AOX+T kinetics are biphasic

as seen in sp.011 wt and sp.011 pREP, (cf. Figure 5.6).

165

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300

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500

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

ro

tein

Qr/Qt

Figure 5.9 Combined uncoupled oxidising pathway kinetics, Qr/Qt vs. vO2, from sp.011 AOX and sp.011

AOX + T mitochondria. Using the NADH regenerating system Qr/Qt and vO2 were measured simultaneously

(NADH titration range 0 – 75 M). sp.011 AOX uncoupled () data were obtained from five mitochondrial

isolations, combining data from nine traces. sp.011 AOX + T state 3 () data were obtained from six

mitochondrial isolations, combining data points from ten traces. The inset shows a representative trace of

uncoupled oxidising pathway kinetics in sp.011 AOX mitochondria. Mitochondrial protein used per

experiment is 0.5-0.7 mg.


AOX and sp.011 AOX+T mitochondria. Clearly the presence of AOX does not change the

relationship. Data points from both sets overlap up until vO2 becomes limiting in sp.011

AOX+T mitochondria. The fact that the relationship between ∆ and vO2 does not change

in the presence of AOX indicates that the functional expression of this protein does not lead

0

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400

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0 0.2 0.4 0.6 0.8 1

vO

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ro

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Qr/Qt

166

0

50

100

150

100 120 140 160 180 200 220

vO

2 n

mo

l O

2 /

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/ m

g p

rote

in


to a disruption of the IMM. It is known that passive proton leak through the bulk

phospholipid bilayer accounts for only a small proportion (between 5 to 25 %) of total

mitochondrial proton leak [184, 244]. It has been hypothesized that the presence of protein

complexes in the IMM can non-specifically increase the permeability of the IMM [245].

Incorporation of AOX into the IMM of S. pombe mitochondria could therefore lead to an

increase in conductivity. Figure 5.10 however shows that the IMM conductivity of S.

pombe mitochondria AOX is the same. Therefore expression of AOX does not affect

IMM conductivity and any differences in respiratory kinetics seen between mitochondria

expressing AOX and mitochondria in which AOX is repressed are therefore likely to be due

to AOX activity.

Figure 5.10 Combined state 2 oxidising pathway kinetics, ∆ vs. vO2, from sp.011 AOX and sp.011 AOX +

T mitochondria. Using the NADH regenerating system ∆ and vO2 were measured simultaneously (NADH

titration range 0 – 75 M). sp.011 AOX () data were obtained from two mitochondrial isolations,

combining data from four traces. sp.011 AOX + T () data were obtained from two mitochondrial

isolations, combining data points from three traces. Mitochondrial protein used per experiment is 0.5-0.7 mg.


AOX and sp.011 AOX + T mitochondria respiring on NADH. There does not appear to be

any difference between both data sets. Interestingly the biphasic pattern is still present in

sp.011 AOX mitochondria and does not look different from the pattern seen in the

167

0

50

100

150

200

250

140 145 150 155 160 165 170 175 180

vO

2 n

mo

l O

2 /

min

/ m

g p

rote

in


mitochondria in which AOX is repressed (or in the wild type mitochondria, cf. Figure 4.7).

As the ∆ is generated by activity of the cytochrome pathway, this indicates that the

biphasic kinetics are a characteristic of cytochrome pathway activity, which corroborates

the hypothesis that the biphasic kinetics are due to bc1 complex activity, see section 4.2.4.

Due to experimental variation it cannot be concluded from the data in Figure 5.11 whether

or not the presence of AOX has an effect on membrane potential.

Figure 5.11 Combined state 3 oxidising pathway kinetics, ∆ vs. vO2, from sp.011 AOX and sp.011 AOX +

T mitochondria. Using the NADH regenerating system ∆ and vO2 were measured simultaneously (NADH

titration range 0 – 75 M). sp.011 AOX () data were obtained from one mitochondrial isolation, combining

data from three traces. sp.011 AOX + T () data were obtained from two mitochondrial isolations,

combining data points from eight traces. Mitochondrial protein used per experiment was 0.5-0.7 mg.

Addition of octyl gallate to sp.011 AOX mitochondria respiring under ADP limited

conditions led to a decrease in vO2 but did not lead to a change in (data not shown).

This can easily be explained by the non-ohmic nature of the vs. vO2 relationship under

ADP limited conditions. Due to the increased conductivity of the IMM at high (see

section 3.1.2) a significant decrease of will only be seen after a disproportional

inhibition of respiration. Given the low capacity of the IMM it only requires the net transfer

of 1 nmol of H+ per mg of protein across the membrane to establish a full [1]. It was

determined in S. pombe mitochondria under ADP limited conditions that a full was

168

established at a rate of ~50 nmol O2 / min / mg protein. If AOX activity under ADP limited

conditions would compete with the cytochrome pathway to such an extent that the steady

state vO2 generated by cytochrome pathway activity would decrease below 50 nmol O2 /

min / mg protein then a decrease in could occur. This however does not occur, the

maximum rate attainable by AOX activity is approximately 80 nmol O2 / min / mg protein,

see Table 5.1. NADH dependent respiration under ADP limited conditions in sp.011 AOX

mitochondria is approximately 200 nmol O2 / min / mg protein and with succinate it is

around 150 nmol O2 / min / mg protein. Therefore in both cases the minimum rate of 50

nmol O2 / min / mg protein (generated through cytochrome pathway activity) in order to

establish a full are easily attained and inhibition of AOX activity will not lead to a

change in under ADP limited conditions. Under state 3 conditions however the

relationship between vs. vO2 in S. pombe mitochondria becomes ohmic after the

biphasic point, i.e. a change in cytochrome pathway activity leads to a proportional change

in , see figures 4.7, 5.5 and 5.11. A linear relationship between and vO2 under state

3 conditions has been demonstrated in potato mitochondria [133, 157]. As a control

experiment for comparison with sp.011 wt respiratory kinetics the relationship between

vs. vO2 under state 3 conditions was determined in potato mitochondria and was found to

be linear, in agreement with the literature, see inset A of Figure 4.7. Under state 3

conditions a decrease in cytochrome pathway activity would lead to a decrease in . If

AOX and cytochrome pathway activity in sp.011 AOX mitochondria would be additive, i.e.

cytochrome pathway activity is not affected by AOX activity, then inhibition of AOX

should not lead to a decrease in . If AOX in sp.011 AOX mitochondria could actively

compete with the cytochrome pathway for reducing equivalents then AOX activity would

lead to a decreased cytochrome pathway activity (compared to the situation where AOX is

absent) and would therefore be proportionally lower. This follows directly from Ohm’s

law:

I = g*E

I: current, g: conductance and E: electrical potential [246].

169

Under state 3 conditions g is constant, indicated by the linear relationship between I

(oxygen consumption rate) and E (). The relationship between I and E is proportional.

When due to AOX activity the electron transfer rate through the cytochrome pathway

would decrease (through competition for reducing equivalents) the oxygen consumption

rate generated through cytochrome pathway activity would decrease and therefore also .

To test this hypothesis the following experiment was done: a state 3 was induced in sp.011

AOX mitochondria respiring on either NADH or succinate by addition of a saturating

amount of ADP (1 mM), when a steady state was reached a saturating amount of octyl

gallate (14 M) was added to completely inhibit AOX. If AOX was actively competing

with the cytochrome pathway then inhibition of this protein would lead to a diversion of

electron flow from the alternative pathway to the cytochrome pathway. The increase in

cytochrome pathway activity would then necessarily lead to an increase in . Figure 5.12

shows a representative experiment where the effect of AOX activity on was

investigated in sp.011 AOX mitochondria respiring on NADH.

Figure 5.12 AOX activity has no effect on . Representative oxygen concentration and traces of sp.011

AOX mitochondria respiring on NADH. Additions NADH (1.8 mM) ADP (1 mM) and octyl gallate (OG, 14

M). See text for details.

0

100

200

300

400

500

600

1.5

1.6

1.7

1.8

1.9

2

2.1

2.2

250 300 350 400 450 500 550 600

ox

yg

en (

nm

ol)

TP

P+

sign

al V

time (s)

TPP+ signal

O2

NADH ADP OG

175

324

249

170

It can be seen that upon addition of a saturating amount of octyl gallate the vO2 rate

decreases significantly, however remains constant. This suggests that in sp.011 AOX

mitochondria the activities of the cytochrome and the alternative pathway are additive.

Comparable results were obtained with succinate as a substrate (data not shown). Based on

these results it was concluded that AOX activity does not have an effect on in S. pombe

mitochondria.

The results shown in figures 5.7 to 5.9 show that under all energetic conditions, in the

presence of AOX, at each value of Qr/Qt the vO2 rates are higher when compared to S.

pombe mitochondria in which AOX is repressed. These results are different from what was

found by Affourtit who found that under state 3 and state 4 conditions, in the presence of

AOX vO2 rates were lower when compared to S. pombe mitochondria in which AOX is

repressed [93]. Under uncoupled conditions no significant difference was found between S.

pombe mitochondria in which AOX was either expressed or repressed [93]. The oxidising

pathway kinetics determined by Affourtit were based on malonate titrations of S. pombe

mitochondria respiring on succinate. In order to investigate the possibility of substrate

dependent differences in oxidising pathway kinetics in S. pombe mitochondria expressing

AOX it was decided to repeat these experiments.

5.2.3 Oxidising pathway kinetics with succinate as substrate

5.2.3.1 Comparing sp.011 AOX and sp.011 AOX + T oxidising pathway kinetics with

succinate as a substrate—With the sp.011 AOX mitochondria it was difficult to obtain

steady state respiratory rates with succinate after additions of malonate during the trace, this

was due to a combination of an effect of malonate24

on the Q-electrode and the long time it

takes for electron flow partition between the cytochrome and alternative pathway to

stabilise after addition of malonate. In fact steady states were rarely reached. This problem

did not occur with NADH as a substrate, presumably because the external NADH

dehydrogenase has a much higher activity than SDH and a steady state electron flow to

24

Upon addition of malonate the Q-electrode signal deflects downwards and recovers slowly, this limits

the amount of data points one can obtain during an experimental trace.

171

both pathways is achieved relatively fast. In order to get around this problem sp.011 AOX

succinate data points were obtained by using ‘standard titrations’ (see section 2.4.4.4).

Steady state Qr/Qt and vO2 values were determined after additions of succinate, ATP and

glutamate respectively (cf. Figure 4.3). This yields three data points which are relatively

spaced apart. When incubated with malonate (thus avoiding effects of malonate addition on

the Q-electrode signal during the trace) the range of these three data points can be shifted to

the left. By combining data sets from several experiments nearly the whole range of Qr/Qt

can be covered. Figure 5.13 shows the relationship between Qr/Qt and vO2 under state 2

and state 4 conditions in sp.011 AOX and sp.011 AOX + T mitochondria respiring on

succinate. In sp.011 AOX mitochondria the vO2 rates at each value of Qr/Qt are higher than

in sp.011 AOX+T mitochondria and both relationships are linear, comparable to what was

seen in the NADH titration experiments, (cf. Figure 5.7). This result was the complete

opposite of what was seen by Affourtit [93] who found that under ADP limited conditions,

at each value of Qr/Qt, in the presence of AOX, vO2 values were lowered when compared

to sp.011 AOX+T mitochondria.

Figure 5.14 shows the relationship between Qr/Qt and vO2 under state 3 conditions

in sp.011 AOX and sp.011 AOX + T mitochondria with succinate as a substrate. In sp.011

AOX mitochondria the vO2 rates at each value of Qr/Qt are higher than in sp.011 AOX+T

mitochondria, similar to what is seen with NADH as a substrate (cf. Figure 5.8). sp.011

AOX+T mitochondria display the familiar biphasic pattern (cf. figures 4.11, 5.4 and 5.8).

The relationship between Qr/Qt vs. vO2 in sp.011 AOX mitochondria is linear (the biphasic

point is masked), similar to what is seen with NADH as a substrate (cf. Figure 5.8). These

results are different from what was found by Affourtit [93]. Under state 3 conditions he

found that at Qr/Qt values higher than 40%, in the presence of AOX, the vO2 rate values

were lower when compared to sp.011 AOX+T mitochondria.

Figure 5.15 shows the relationship between Qr/Qt and vO2 under uncoupled

conditions in sp.011 AOX and sp.011 AOX + T mitochondria respiring on succinate. In

sp.011 AOX mitochondria the vO2 rates at each value of Qr/Qt are higher than in sp.011

AOX+T mitochondria, similar to what is seen with NADH as a substrate (cf. Figure 5.9).

sp.011 AOX+T mitochondria display the familiar biphasic pattern (cf. figures 4.12, 5.6 and

5.9). The relationship between Qr/Qt vs. vO2 in sp.011 AOX mitochondria is linear (the

172

biphasic point is masked), similar to what is seen with NADH as a substrate (cf. Figure

5.9). These results are different from what was found by Affourtit [93]. Under uncoupled

conditions he found that the oxidising pathway kinetics of sp.011 AOX and sp.011 AOX+T

mitochondria were virtually identical.

Figure 5.13 Combined state 2 and state 4 oxidising pathway kinetics, Qr/Qt vs. vO2, from sp.011 AOX and

sp.011 AOX + T mitochondria. Succinate-dependent respiration was titrated with malonate with sp.011

AOX+T mitochondria. Standard titrations were used with both sp.011 AOX and sp.011 AOX+T

mitochondria. Qr/Qt and vO2 were measured simultaneously. sp.011 AOX state 2 () and state 4 (∆) data

were obtained from four and four mitochondrial isolations respectively, combining data points from six and

five traces respectively. sp.011 AOX + T state 2 () and state 4 () malonate titration data were obtained

from one and two mitochondrial isolations respectively, combining data points from two and five traces

respectively. sp.011 AOX+T standard data points were obtained from five and four mitochondrial isolations

respectively, combining data points from six and five traces respectively. Mitochondrial protein used per

experiment is 0.5-0.7 mg.

0

50

100

150

200

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

ro

tein

Qr/Qt

173

Figure 5.14 Combined state 3 oxidising pathway kinetics, Qr/Qt vs. vO2, from sp.011 AOX and sp.011 AOX

+ T mitochondria. Succinate-dependent respiration was titrated with malonate with sp.011 AOX+T

mitochondria. Standard titrations were used with sp.011 AOX and sp.011 AOX+T mitochondria. Qr/Qt and

vO2 were measured simultaneously. sp.011 AOX data () were obtained from six mitochondrial isolations,

combining data points from eleven traces. sp.011 AOX + T data () were obtained from three

mitochondrial isolations, combining data points from six traces (malonate titrations) and from five

mitochondrial isolations, combining six traces (standard traces). Mitochondrial protein used per experiment is

0.5-0.7 mg.

To investigate the cause(s) of the differences between the results of Affourtit [93] and the

results obtained in this study several possibilities were considered. The method of isolation

was different and it was known that the mitochondria used in Affourtit’s study were of

different quality. The RCR value of sp.011 AOX+T mitochondria respiring on NADH

(uncoupled / state 2) in this study is about 5 (Table 5.3). The equivalent RCR value in

Affourtit’s study is estimated by this author to be around 2 (cf. Figure 2A in [93]). More

importantly though, the reaction medium which was used in Affourtit’s study for the

measurement of Qr/Qt and vO2 had a lower osmolarity from what is customarily used in

yeast studies [104, 105, 160, 240, 247]. The reaction medium used in this study (see section

0

50

100

150

200

250

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

ro

tein

Qr/Qt

174

2.1.1.7) contains a mannitol concentration of 0.65 M whereas the medium used by Affourtit

(potato reaction medium, see section 2.1.3.2) contains a mannitol concentration of 0.3 M. It

has been reported that lowering of the osmolarity leads to a decrease in vO2 rates in S.

pombe mitochondria [105]. To further investigate this, the relationship between Qr/Qt and

vO2 under state 3 conditions was determined in sp.011 AOX mitochondria in potato

reaction medium (0.3 M mannitol) and yeast reaction medium (0.65 M mannitol), see

Figure 5.16. The oxidising pathway kinetics in the low osmotic medium level off at high

values of Qr/Qt. These results suggest that a difference in osmotic strength of the reaction

medium affects S. pombe respiratory kinetics.

Figure 5.15 Combined uncoupled oxidising pathway kinetics, Qr/Qt vs. vO2, from sp.011 AOX and sp.011

AOX + T mitochondria. Succinate-dependent respiration was titrated with malonate with sp.011 AOX+T

mitochondria. Standard titrations were used with sp.011 AOX mitochondria. Qr/Qt and vO2 were measured

simultaneously. sp.011 AOX data () were obtained from three mitochondrial isolations, combining data

points from seven traces . sp.011 AOX + T data () were obtained from two mitochondrial isolations,

combining data from five traces (malonate titration data) and from three mitochondrial isolations, combining

data from three traces (standard traces). Mitochondrial protein used per experiment is 0.5-0.7 mg.

0

50

100

150

200

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

rote

in

Qr/Qt

175

Figure 5.16 Combined state 3 oxidising pathway kinetics, Qr/Qt vs. vO2, from sp.011 AOX mitochondria in

reaction medium containing 0.3 M mannitol () or 0.65 M mannitol (). The oxidising pathway kinetics

were obtained using the NADH regeneration method (NADH titration range 0 – 75 M). State 3 conditions

were induced by pre-incubation with ADP (1 mM). The high osmolarity (0.65 M mannitol) data were

obtained from two mitochondrial isolations, data points taken from three traces. The low osmolarity (0.3 M

mannitol) data were obtained from two mitochondrial isolations, data points taken from four traces.

Mitochondrial protein used per experiment is 0.5-0.7 mg.

At the time when the research on S. pombe mitochondria was conducted by Affourtit it was

unknown that S. pombe mitochondria display ethanol dependant respiration as was

demonstrated later by Crichton [26]. Addition of small amounts of pure ethanol (as low as 1

l) will result in a significant oxygen consumption rate. Many of the substances added by

Affourtit during his experiments were dissolved in ethanol, Q1 which was present in all

experiments being one of them. The addition of ethanol to S. pombe mitochondria in

solution will lead to ADH activity, (see section 1.3.1) which results in matrix NADH

generation. Possibly the respiratory kinetics determined by Affourtit [93] with succinate as

a substrate were a result of combined SDH and internal NADH dehydrogenase activities.

Respiratory kinetics determined with NADH as a substrate were possibly a result of

combined external and internal NADH dehydrogenase activities.

0

50

100

150

200

250

300

350

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

rote

in

Qr/Qt

176

5.2.4 Oxidising pathway kinetics in sp.011 AOX mitochondria

Results so far show that independent of substrate in S. pombe mitochondria expressing

AOX, under all energetic conditions, at each value of Qr/Qt, the vO2 rates are higher when

compared to sp.011 AOX+T mitochondria. Also, in the presence of AOX the biphasic

kinetics under state 3 and uncoupled conditions are no longer seen and the relationship

between Qr/Qt and vO2 becomes linear (the biphasic pattern is masked). Table 5.2 shows

that steady state oxygen consumption rates with succinate as a substrate under all energetic

conditions are lower than with NADH as a substrate. This could be due to SDH activity

becoming limiting. For instance, in figures 4.11 and 4.12 the cytochrome pathway kinetics

with either succinate or NADH as a substrate are clearly the same. With NADH as a

substrate however the Q-pool can be reduced to a higher degree than with succinate, which

results in higher maximum vO2 rates attainable with NADH as a substrate. sp.011 AOX

oxidising pathway kinetics under various energetic conditions with either succinate or

NADH as a substrate were plotted together to see if there were any substrate dependent

differences in kinetics.

5.2.4.1 Comparing sp.011 AOX oxidising pathway kinetics with either NADH or succinate

as a substrate—Figure 5.17 shows the relationship between Qr/Qt and vO2 under state 2

and state 4 conditions in sp.011 AOX mitochondria respiring on either NADH or succinate.

It can be appreciated that at high Qr/Qt (~60%) and upwards the vO2 values attained with

NADH as a substrate are higher than with succinate. Figure 5.18 shows the relationship

between Qr/Qt and vO2 under state 3 conditions in sp.011 AOX mitochondria respiring on

either NADH or succinate. It is clear that from Qr/Qt values around 40% and upwards with

NADH as a substrate the vO2 rates at each value of Qr/Qt are higher than with succinate as

a substrate. Figure 5.19 shows the relationship between Qr/Qt and vO2 under uncoupled

conditions in sp.011 AOX mitochondria respiring on either NADH or succinate. The data

suggests that from Qr/Qt values around 40% Qr/Qt with NADH as a substrate the vO2 rates

at each value of Qr/Qt are higher than with succinate.

177

Figure 5.17 Comparing state 2 and state 4 oxidising pathway kinetics in sp.011 AOX mitochondria respiring

on either NADH or succinate. Succinate state 2 () and state 4 () were taken from Figure 5.13. NADH

state 2 data () were taken from Figure 5.7.

Figure 5.18 Comparing state 3 oxidising pathway kinetics in sp.011 AOX mitochondria respiring on either

NADH or succinate. Succinate () data were taken from Figure 5.14. NADH () data were taken from

Figure 5.8.

0

50

100

150

200

250

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

ro

tein

Qr/Qt

0

50

100

150

200

250

300

350

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

ro

tein

Qr/Qt

178

Figure 5.19 Comparing uncoupled oxidising pathway kinetics in sp.011 AOX mitochondria respiring on

either NADH or succinate. Succinate () data were taken from Figure 5.15. NADH () data were taken from

Figure 5.9.

The results shown in figures 5.17-5.19 indicate that there are substrate dependent

differences under all energetic conditions in sp.011 AOX mitochondria respiring on either

succinate or NADH. It was shown in chapter 4 (cf. figures 4.9-4.12) that wild type S.

pombe mitochondria do not display any substrate dependent differences in cytochrome

pathway kinetics. The results presented in section 5.2.2.1 (cf. figures 5.2-5.6) show that

transformation of S. pombe mitochondria does not change its respiratory kinetics. However

the sp.011 wt and sp.011 pREP respiratory kinetics are somewhat different from the sp.011

AOX+T and sp.011 AOX (incubated in the presence of OG) mitochondria as reflected in

differences between vO2 rates and RCR values, see section 5.2.1. sp.011 wt and sp.011

pREP mitochondria may not be the proper control group. To verify whether or not the

substrate dependent differences observed in sp.011 AOX mitochondria are due to altered

cytochrome pathway kinetics induced by the transformation with a vector coding for AOX

the cytochrome pathway kinetics with either succinate or NADH as a substrate from sp.011

AOX+T (AOX expression repressed) were compared.

0

100

200

300

400

500

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

ro

tein

Qr/Qt

179

5.2.5 Cytochrome pathway kinetics in sp.011 AOX+T mitochondria

5.2.5.1 Comparing sp.011 AOX + T cytochrome pathway kinetics with either NADH or

succinate as a substrate—Figure 5.20 shows the relationship between Qr/Qt and vO2 under

state 2 and state 4 conditions in sp.011 AOX + T mitochondria respiring on either succinate

or NADH. Data points obtained with succinate overlap with those obtained with NADH.

This suggests that under ADP limited conditions in sp.011 AOX+T mitochondria there are

no substrate dependent differences in cytochrome pathway kinetics.

Figure 5.20 Comparing state 2 and state 4 cytochrome pathway kinetics in sp.011 AOX + T respiring on

either NADH or succinate. Succinate state 2 (, ) and state 4 (, ) data were taken from Figure 5.13.

NADH state 2 () and state 4 (∆) data were taken from Figure 5.7.

Figure 5.21 shows the relationship between Qr/Qt and vO2 under state 3 conditions in

sp.011 AOX + T mitochondria respiring on either succinate or NADH. No apparent

substrate differences can be seen in cytochrome pathway kinetics. Figure 5.22 shows the

relationship between Qr/Qt and vO2 under uncoupled conditions in sp.011 AOX + T

mitochondria respiring on either succinate or NADH. No apparent substrate dependent

differences can be seen in the cytochrome pathway kinetics.

0

20

40

60

80

100

120

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

ro

tein

Qr/Qt

180

Figure 5.21 Comparing state 3 cytochrome pathway kinetics in sp.011 AOX + T respiring on either NADH or

succinate. Succinate (, ) data were taken from Figure 5.14. NADH () data were taken from Figure 5.8.

Figure 5.22 Comparing uncoupled cytochrome pathway kinetics in sp.011 AOX + T mitochondria respiring

on either NADH or succinate. Succinate (, ) data were taken from Figure 5.15. NADH () data were taken

from Figure 5.9.

0

50

100

150

200

250

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

rote

in

Qr/Qt

0

50

100

150

200

250

300

350

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

rote

in

Qr/Qt

181

It can be concluded from the results shown in figures 5.20-5.22 that there are no substrate

dependent differences in sp.011 AOX+T cytochrome pathway kinetics. This suggests that

the substrate dependent differences observed in sp.011 AOX mitochondria are not due to

transformation of S. pombe cells with a vector containing the S. guttatum AOX gene.

Equally also it can be concluded from figures 5.20-5.22 that inclusion of thiamine in the

growth medium does not affect cytochrome pathway kinetics. Since the substrate dependent

differences in oxidising pathway kinetics in sp.011 AOX mitochondria cannot be attributed

to cytochrome pathway characteristics the most likely cause would be a substrate dependent

difference in alternative pathway kinetics.

5.2.6 Alternative pathway kinetics in sp.011 AOX mitochondria

5.2.6.1 Comparing sp.011 AOX alternative pathway kinetics with either NADH or

succinate as a substrate—To investigate substrate dependent differences in alternative

pathway kinetics, sp.011 AOX mitochondria were incubated with antimycin A to inhibit the

cytochrome pathway [1]. Using either the NADH regenerating system or the malonate

titration method alternative pathway kinetics were determined with either NADH or

succinate as a substrate. For this experiment four different mitochondrial isolations were

used and per isolation several traces of both NADH and succinate dependent respiration

were determined. Unfortunately there was quite some variation in vO2 rates between

isolations and combining the data would give the impression that with succinate as a

substrate higher vO2 rates are obtained at each value of Qr/Qt in comparison to NADH

kinetics. However, when analysing the data per day it was obvious that there are no

substrate dependent differences in alternative pathway kinetics. Figure 5.23 shows

representative data from one of the experimental days. From these results it was concluded

that the substrate dependent differences as seen in sp.011 AOX oxidising pathway kinetics

are not due to underlying substrate dependent alternative pathway kinetics.

182

0

20

40

60

80

100

120

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

rote

in

Qr/Qt

Figure 5.23 Comparing alternative pathway activity in sp.011 AOX mitochondria respiring on either

succinate or NADH. Qr/Qt and vO2 were determined simultaneously, antimycin A was incubated (2.5 µL of 1

mg/ml AA). Succinate data points () taken from two traces, NADH data points () taken from three traces.

Mitochondrial protein used is 0.5 mg.

The antimycin-insensitive respiration rates displayed by sp.011 AOX mitochondria

respiring on either succinate or NADH are comparable (cf. Table 5.2). In many plants

antimycin-insensitive respiration with succinate is relatively high when compared with

NADH dependent respiration [74]; this is due to the fact that with exogenous NADH as

substrate no pyruvate is generated which can activate AOX [79, 248]. NADH respiratory

rates being comparable to succinate respiratory rates in sp.011 AOX mitochondria,

incubated in the presence of antimycin A, indicate that AOX expressed in S. pombe

mitochondria is constitutively active, as has been demonstrated previously [70].

The results so far show that S. pombe mitochondria heterologously expressing a

plant AOX display substrate dependent oxidising pathway kinetics which cannot be

explained in terms of cytochrome pathway or alternative pathway kinetics. The substrate

dependent differences in oxidising pathway kinetics may be a particularity of the

expression system used in this study. For comparison, oxidising pathway kinetics with

either succinate or NADH as a substrate were determined in isolated mitochondria from

Arum maculatum, a thermogenic plant species [249] which naturally expresses AOX [250].

183

5.2.7 Oxidising pathway kinetics in Arum maculatum mitochondria

5.2.7.1 Investigating substrate dependent differences in oxidising pathway kinetics in Arum

maculatum mitochondria—Arum maculatum is, just like Sauromatum guttatum [251] a

thermogenic plant species. During the development of thermogenesis A. maculatum

spadices pass through a recognizable sequence of developmental stages (termed -) which

results in a rapid rise in respiration that is responsible for a period of heat production during

pollination [88]. Heat production can lead to a temperature difference with the environment

of about 15 C [251], the aim of generating heat being to volatilise aromatic compounds to

attract pollinators [66]. In A. maculatum mitochondria isolated during the -stage the

alternative pathway is barely active, whereas during the / stage alternative pathway

activity is much more pronounced. During the -stage virtually all respiration is due to

alternative pathway activity [88]. In this study arum spadices were collected in local

Sussex woods during the / stage in order to investigate possible substrate dependent

differences in oxidising pathway kinetics in an organism naturally expressing both the

cytochrome and the alternative pathway. When arum spadices are cold stored (4 C)

overnight, prior to mitochondrial isolation both cytochrome pathway and SDH activities are

increased, whereas alternative pathway activity remains unaltered [88]. Arum mitochondria

were isolated from freshly picked or cold stored spadices (see section 2.1.4 for isolation

protocol). In S. pombe mitochondria expressing AOX the activity of the alternative pathway

is considerable given the fact that under all energetic conditions, at each value of Qr/Qt the

respiratory rates in mitochondria expressing AOX are higher than in mitochondria in which

AOX is repressed, see figures 5.7-5.9 and 5.13-5.15. However, under uncoupled conditions

most of the respiratory activity is due to cytochrome pathway activity since the maximum

alternative pathway activity is limited to 80-100 nmol O2 / min / mg protein and the total

respiration rate in uncoupled sp.011 AOX mitochondria is around 530 nmol O2 / min / mg

protein (rate with NADH as a substrate). It appears that in fresh A. maculatum mitochondria

in the / stage the alternative pathway has a larger contribution to overall respiration rate

than in S. pombe mitochondria. By cold storing the spadices the cytochrome pathway in A.

maculatum mitochondria should become more active, which would increase its contribution

to overall respiration rate, a situation more comparable to the yeast expression system.

184

0

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400

600

800

1000

1200

0 0.2 0.4 0.6 0.8 1

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2 /

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/ m

g p

rote

in

Qr/Qt

In both types of arum mitochondria (‘fresh’ and ‘cold stored’) both pathways were active.

When incubated with OG and CCCP a limited respiration rate (cytochrome pathway

activity) was measured. In the absence of OG and presence of CCCP the respiration rate

was 3 to 4 times higher, demonstrating that the alternative pathway was active.

Figure 5.24 shows the relationships between Qr/Qt and vO2 under uncoupled

conditions in A. maculatum mitochondria respiring either on succinate or NADH. Data

obtained from mitochondria isolated from freshly picked or cold stored spadices are

combined. Although the data shows considerable variation it is clear that there are no

substrate dependent differences in oxidising pathway kinetics. Equally also it is apparent

that there are no differences between oxidising pathway kinetics of mitochondria isolated

from fresh spadices or cold stored ones.

Figure 5.24 Comparing uncoupled oxidising pathway kinetics in Arum maculatum mitochondria respiring on

either succinate or NADH. Qr/Qt and vO2 were determined simultaneously. Succinate fresh Arum data ()

and cold stored Arum data (▲) were obtained from three and two mitochondrial isolations respectively,

combining data points from six and five traces respectively (succinate 9 mM, ATP 0.2 mM). NADH fresh

arum data () and cold stored arum data (∆) were obtained from four and two mitochondrial isolations

respectively, combining data points from eight and six traces respectively. Mitochondrial protein used per

experiment 0.1 – 0.3 mg. NADH and succinate data points were obtained using the malonate titration and the

NADH regenerating method. NADH titration range (0-50 M) malonate titration range (0-4 mM)

185

DOES THE ALTERNATIVE PATHWAY IN sp.011 AOX MITOCHONDRIA

SERVE AS AN OVERFLOW?

It has been a long held belief that the alternative pathway in AOX expressing mitochondria

served as an overflow pathway under conditions where the cytochrome pathway is either

saturated or inhibited [79]. It was originally found by Bahr and Bonner [252, 253] that upon

addition of hydroxamic acids (inhibitors of AOX) switching of electron flow from the

alternative pathway to the cytochrome pathway did not occur. Later studies showed that

AOX only becomes engaged at relatively high Qr/Qt values [75, 78, 86] corroborating the

overflow hypothesis. Results by Wilson [237] and Hoefnagel et al. [79] successfully

challenged the overflow hypothesis by demonstrating that diversion of electron flow from

the alternative pathway to the cytochrome pathway can occur. Also, the overflow

hypothesis requires the cytochrome pathway to be saturated before electrons can be

diverted to the alternative pathway, it was however demonstrated by van den Bergen et al.

that the cytochrome pathway never attains a saturated rate, even at high levels of Q-

reduction [76]. Assessment of the contribution of AOX to overall respiration rate by

inhibition of AOX can lead to an underestimation, since electron flow can be diverted from

the alternative pathway to the cytochrome pathway, possibly giving the impression that

AOX was not engaged prior to addition of inhibitor. The alternative and cytochrome

pathway share the same substrate (QH2), inhibition of one pathway will perturb the Qr/Qt

ratio which will affect the activity of the uninhibited pathway [148]. To give an example, a

naive approach to estimate the contribution of AOX to total respiration would be to

measure total respiration, inhibit the alternative pathway (with octyl gallate or SHAM for

instance), measure the residual respiration and then subtract these two rates from each

other. Inhibition of AOX could lead to an increase in Qr/Qt, this will lead to an increase in

cytochrome pathway activity. The rate thus measured does not reflect cytochrome pathway

activity prior to addition of AOX inhibitor and subtracting this rate from total respiration

rate will give an underestimation of AOX contribution to the total respiration rate.

186

It is now firmly established that in plant mitochondria AOX does not function simply as an

overflow pathway. The situation in plants however cannot simply be extrapolated to our

yeast expression system. The alternative oxidase is a heterologous protein in the electron

transfer chain of our expression system and it might be the case that in our system there is a

preference for the cytochrome pathway over the alternative pathway.

Bahr and Bonner [252] introduced an inhibitor titration technique which is used to

provide an estimation of the contribution of AOX to respiration in the absence of inhibitors.

Oxygen consumption is measured during an inhibitor titration of AOX activity (with octyl

gallate for instance) in the presence and absence of a cytochrome pathway inhibitor.

Plotting of these data sets against each other yields so called plots [252], which gives a

measure of AOX engagement during respiration in the absence of AOX inhibitor. If the

relationship between the two sets is linear it can be concluded that the electron flux through

the cytochrome pathway is independent from the flux through the alternative pathway. A

‘break’ in this relationship indicates that electron flow is diverted from the alternative

pathway to the cytochrome pathway. Figure 5.25 shows a plot where NADH dependent

respiration was titrated with OG in the presence and absence of antimycin A. Both datasets

were plotted against each other and it can be seen that there is a breakpoint in the

relationship. This indicates that during the initial additions of OG electron flow was

diverted to the cytochrome pathway and total respiration is seen to be resistant against OG

inhibition. When the cytochrome pathway is saturated further addition of OG leads to a

decrease in total respiration as no more electrons can be diverted from the alternative

pathway to the cytochrome pathway. Figure 5.26 shows a similar experiment where sp.011

AOX mitochondria were titrated with antimycin A in the presence and absence of octyl

gallate. This experiment indicates that upon inhibition of the cytochrome pathway electron

flow can be diverted to the alternative pathway, i.e. the alternative pathway is not saturated.

Similar results were obtained with succinate as a substrate (data not shown).

187

Figure 5.25 Alternative pathway plot. Two octyl gallate titration experiments were performed on sp.011

AOX mitochondria in the presence and absence of antimycin A. NADH (1.8 mM) antimycin A (2 M) octyl

gallate titration range (0 – 1 M). Data from one mitochondrial isolation. Amount of protein used: 0.5 mg.

Figure 5.26 cytochrome pathway plot. Two antimycin A titration experiments were performed on sp.011

AOX mitochondria in the presence and absence of octyl gallate. NADH (1.8 mM) octyl gallate (2.3 M)

antimycin A titration range (0 – 40 nM). Data obtained from one mitochondrial isolation. Amount of protein

used: 0.5 mg.

0

50

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200

0 20 40 60 80 100 120

OG

tit

rati

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g p

rote

in

OG titration with AA incubatednmol O

2 / min / mg protein

0

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0 20 40 60 80 100

AA

tit

rati

on

AA titration with OG incubatednmol O

2 / min / mg protein

188

The results in figures 5.25 and 5.26 indicate that under ADP limited conditions neither

pathway is saturated and that electron flow can be diverted from one pathway to the other.

These results are in agreement with work done on plants naturally expressing AOX [79].

The main reason why the overflow hypothesis was not successfully challenged for almost

two decades is due to the fact that only relatively recently it became clear that AOX is

partially inactive in isolated plant mitochondria. Upon addition of pyruvate AOX is seen to

become active at lower values of Qr/Qt [79]. In soybean cotyledon mitochondria

intramitochondrial pyruvate is generated during succinate oxidation, but at low

concentrations. Under these conditions inhibitor titrations may fail to demonstrate the

ability of AOX to compete with the cytochrome pathway. In S. pombe mitochondria

expressing the S. guttatum AOX it was found that AOX was constitutively active [70], a

situation comparable to pyruvate activated AOX in plant mitochondria. The results in

figures 5.25 and 5.26 agree well the work done by Hoefnagel et al. on fully activated AOX

in soy bean cotyledon mitochondria [79]. In S. pombe mitochondria with either NADH or

succinate as a substrate, under all energetic conditions, the respiratory rate at each value of

Qr/Qt is higher in the presence than in the absence of AOX. Cytochrome pathway activity

with NADH is higher than with succinate under state 3 and uncoupled conditions.

Therefore with succinate as a substrate, under state 3 and uncoupled conditions, the

cytochrome pathway clearly is not saturated. Since AOX is engaged under all energetic

conditions, that means that in S. pombe mitochondria, the cytochrome pathway does not

need to be saturated in order for the alternative pathway to become engaged.

189

0

50

100

150

200

250

0 0.2 0.4 0.6 0.8 1

vO2 n

mol

O2 /

min

/ m

g p

rote

in

Qr/Qt

Does the expression of AOX lead to a change in Q-pool behaviour?

Classical pool behaviour [23, 24] was found in S. pombe mitochondria (see section 4.2.3.1

and [26]). In sp.011 AOX mitochondria an extra protein complex is introduced into the

ETC. The IMM of mitochondria has a very high protein content25

when compared to other

membranes. It was already found that insertion of AOX had no disruptive effects and the

IMM conductivity was unchanged (cf. Figure 5.10). Although membrane permeability was

not affected, it is possible that introducing an extra respiratory complex to a membrane

which already is relatively protein packed may affect the fluidity with the membrane phase

becoming more viscous. Perhaps the substrate dependent differences in sp.011 AOX

oxidising pathway kinetics are due to a change in ETC organisation with a concomitant

departure of Q-pool behaviour. In order to investigate this reducing pathway kinetics were

determined with succinate as a substrate under various energetic conditions, see Figure

5.27.

Figure 5.27 sp.011 AOX reducing pathway kinetics with succinate as a substrate. Reducing pathway kinetics

were determined under state 2 () and uncoupled () conditions. State 2 conditions: Succinate (9 mM) was

added to sp.011 AOX mitochondria incubated in the presence of ATP (0.2 mM) and glutamate (9 mM).

Uncoupled conditions: like state 2 but with CCCP (2 M) incubated. Reducing pathway kinetics were

obtained by titrating with antimycin A (0 – 40 nM). State 2 and uncoupled data were obtained from one

mitochondrial isolation combining data points from 3 and 4 experimental traces respectively. Amount of

mitochondrial protein used: 0.7 mg.

25

The IMM typically consists of 50% integral protein, 25% peripheral protein and 25% lipid [1].

190

It can be seen that like in the wild type mitochondria (cf. Figure 4.13) there is a clear

difference in reducing pathway kinetics depending on energy state reflecting SDH

deactivation. Homogenous pool behaviour can be demonstrated by showing a sigmoidal

relationship between respiration and increasing antimycin A concentration (which is

reflected in increasing Qr/Qt), a so called resistance to inhibition (cf. Figure 1.9). It is

difficult to distinguish between a linear and a sigmoidal relationship between respiration

rate and Qr/Qt from Figure 5.27. Investigation of individual traces however clearly shows a

sigmoidal relationship, see Figure 5.28, which indicates that S. pombe mitochondria

expressing AOX display pool behaviour.

Figure 5.28 Antimycin A titration of sp.011 AOX mitochondria , representative state 2 trace

taken from Figure 5.26.

It was concluded that functional expression of the alternative oxidase in S. pombe

mitochondria does not affect Q-pool behaviour and the dependency of the SDH reducing

pathway kinetics on energy status of the IMM is comparable to the wild type mitochondria.

50

100

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200

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Qr/Qt

191

5.3 DISCUSSION

5.3.1 Differences between the various S. pombe mitochondria used—In this study the

effects of a functionally expressed plant alternative oxidase in S. pombe mitochondria on

respiratory kinetics were investigated. From the mitochondria used three types were

isolated from transformed S. pombe cells. sp.011 AOX, mitochondria expressing AOX.

sp.011 AOX+T, mitochondria isolated from S. pombe cells transformed with a plasmid

containing the gene for AOX but grown in the presence of thiamine, which represses AOX

expression. sp.011 pREP, mitochondria isolated from S. pombe cells transformed with a

plasmid lacking the gene encoding AOX.

It was a priori expected that the respiratory characteristics of sp.011 wt, sp.011

AOX+T and sp.011 pREP mitochondria would be comparable given that these

mitochondria all lack the alternative pathway. It was found however that based on

respiratory rates and RCR values (see Tables 5.2 and 5.3) that sp.011 wt and sp.011 pREP

mitochondria are similar, with comparable respiratory rates and RCR values whereas the

sp.011 AOX+T mitochondria displayed lower respiratory rates and RCR values. When

incubated with OG alternative pathway activity in sp.011 AOX mitochondria is inhibited

and the respiratory characteristics should be similar to S. pombe mitochondria lacking the

alternative pathway. It was found that sp.011 AOX (incubated with OG) respiratory rates

and RCR values were lower than those of sp.011 wt and sp.011 pREP mitochondria, but

were comparable with values found in sp.011 AOX+T mitochondria. Based on this the S.

pombe mitochondria used in this study were treated as two groups:

sp.011 wt and sp.011 pREP mitochondria (which display relatively high respiratory rates

and RCR values) and sp.011 AOX and sp.011 AOX+T mitochondria (displaying somewhat

lower respiratory rates and RCR values). It can be seen in Table 5.1 that each type of

mitochondria is isolated from yeast cultures with slightly different growth conditions.

sp.011 wt cells are grown in the presence of leucine. This however cannot cause any

specific differences in respiratory kinetics since the respiratory kinetics of sp.011 wt and

sp.011 pREP (no leucine during growth) are comparable. The act of transformation itself

cannot be the cause for a difference in kinetics since the respiratory kinetics of sp.011 wt

(not transformed) mitochondria are comparable to those of sp.011 pREP mitochondria.

192

The presence of thiamine cannot be the cause of differences in respiratory kinetics since the

respiratory kinetics of sp.011 AOX+T mitochondria are comparable of those of sp.011

AOX (incubated with OG) mitochondria. The only thing that sp.011 AOX and sp.011

AOX+T mitochondria have in common in which they are different from sp.011 wt and

sp.011 pREP is the fact that both types of mitochondria are isolated from S. pombe cells

transformed with the plasmid containing the gene encoding for AOX. It is unclear in what

way the presence of a plasmid containing the gene for S. guttatum AOX can affect the

respiratory kinetics of S. pombe mitochondria. Although the differences between the two

groups of S. pombe mitochondria cannot be satisfactorily explained it is clear that the

sp.011 AOX+T mitochondria are a proper control group for comparison with sp.011 AOX

mitochondria. When sp.011 AOX mitochondria are incubated with OG, inhibiting the

alternative pathway, its respiratory kinetics are similar to those of the sp.011 AOX+T

mitochondria.

5.3.2 Does transformation of S. pombe mitochondria lead to changes in respiratory

kinetics?—It could be argued that transformation of yeast cells may cause a change in

mitochondrial respiratory kinetics. Figures 5.2-5.6 indicate that the respiratory kinetics of

transformed S. pombe mitochondria (sp.011 pREP) are not significantly different from

those of the wild type mitochondria (sp.011 wt). Under ADP limited conditions the vO2 vs.

Qr/Qt relationship is linear (Figure 5.2) and under state 3 and uncoupled conditions the vO2

vs. Qr/Qt relationships display a biphasic pattern, in agreement with what was seen in the

wild type mitochondria. Also under ADP limited conditions the vO2 vs. relationships of

sp.011 pREP and sp.011 wt mitochondria overlap (Figure 5.3), which suggests that the

IMM conductivity is not affected by the transformation.

5.3.3 Comparing sp.011 AOX and sp.011 AOX+T oxidising pathway kinetics with NADH

as a substrate—In a previous study done in this laboratory oxidising pathway kinetics were

determined in sp.011 AOX and sp.011 AOX+T mitochondria by titrating succinate

dependent respiration with malonate [93]. Succinate was chosen as substrate since NADH

at saturating concentrations interferes strongly with the Q-electrode. The amount of data

obtained in that study was somewhat limited and the mitochondria used were of relatively

193

poor quality. Hoefnagel et al. [146] demonstrated that NADH can be used in combination

with the Q-electrode to determine oxidising pathway kinetics by using an NADH

regenerating system that requires addition of sub-saturating amounts of NADH; which only

has a limited effect on the Q-electrode signal, that can be corrected for, see section 2.4.4.4.

This method allows for probing of the relationship between vO2 and Qr/Qt covering the

whole range of Qr/Qt (which is more difficult when titrating succinate dependent

respiration with malonate). It was decided to determine the oxidising pathway kinetics in

sp.011 AOX and sp.011 AOX+T mitochondria under various energetic conditions using the

NADH regenerating system, see figures 5.7-5.11. It was found that under all energetic

conditions, in the presence of AOX, the vO2 rates, at each value of Qr/Qt are higher when

compared to sp.011 AOX+T mitochondria with NADH as a substrate. These results were

different from Affourtit’s study [93] who reported that under state 3 and state 4 conditions,

in the presence of AOX, vO2 rates were lowered, whereas under uncoupled conditions

expression of AOX did not seem to affect oxidising pathway kinetics (with succinate as a

substrate).

Under ADP limited conditions the dependency of respiration on Qr/Qt was

proportional for both sp.011 AOX and sp.011 AOX+T mitochondria, similar to what is

seen in sp.011 pREP and sp.011 wt mitochondria (see Figure 5.2). Under state 3 and

uncoupled conditions the sp.011 AOX+T mitochondria display a clear biphasic relationship

between vO2 and Qr/Qt (see figures 5.8 and 5.9), similar to what is seen in sp.011 pREP

and sp.011 wt mitochondria (see figures 5.4 and 5.6). Interestingly the relationship between

Qr/Qt and vO2 under state 3 and uncoupled conditions in sp.011 AOX mitochondria is

linear (see figures 5.8 and 5.9). When scrutinizing individual traces, it is clear that the

biphasic pattern is still present under uncoupled conditions although the biphasic point is

shifted to the left. Apparently the introduction of an extra oxidising pathway leads to

masking of the biphasic pattern under state 3 and uncoupled conditions. This result

corroborates the hypothesis that the biphasic pattern seen in S. pombe mitochondria is a

characteristic of the cytochrome pathway. In chapter 4 (see section 4.2.5.2) it was already

indicated that addition of an extra terminal oxidase changed the oxidising pathway kinetics.

If the biphasic pattern was a characteristic of the dehydrogenase kinetics then the

introduction of AOX should not change the shape of the Qr/Qt vs. vO2 relationship.

194

Incorporation of the alternative oxidase into the IMM of S. pombe mitochondria might have

a disruptive effect, uncoupling the mitochondria. The increase in vO2 observed in sp.011

AOX mitochondria could be due to uncoupling. However, it was shown that under ADP

limited conditions the relationship between vs. vO2 in sp.011 AOX and sp.011 AOX+T

mitochondria is the same (see Figure 5.10) which suggests that the conductivity of the IMM

in both types of mitochondria is the same and that incorporation of an extra protein

complex into the IMM did not lead to a change in proton permeability. Under state 3

conditions, in both sp.011 AOX and sp.011 AOX+T the vs. vO2 relationship is biphasic

(Figure 5.11), similar to what is seen in sp.011 pREP and sp.011 wt (see Figure 5.5). Since

AOX is non-protonmotive, this indicates that the biphasic pattern is a characteristic of the

cytochrome pathway.

5.3.4 Does AOX activity affect in S. pombe mitochondria?—Due to the non-ohmic

relationship between vs. vO2 under ADP limited conditions activity of AOX will not

have an effect on , see section 5.2.2.2. Under state 3 conditions however the relationship

between vs. vO2 in the region following the inflection point of the biphasic pattern is

proportional, i.e. a change in cytochrome pathway activity leads directly to a change in .

If both pathways were to compete with each other for reducing equivalents, then AOX

activity could lead to a decreased electron flow though the cytochrome pathway. If under

state 3 conditions in sp.011 AOX mitochondria the rate of electron transfer through the

cytochrome pathway would be lower than in sp.011 AOX+T mitochondria, then because of

the proportional relationship between vO2 and the maximum attainable in sp.011

AOX mitochondria should be lower than in sp.011 AOX+T mitochondria. Unfortunately,

due to experimental variation this cannot be demonstrated with the available NADH

titration data, see Figure 5.11. To investigate the possible effect of AOX expression on

under state 3 conditions a different approach was used. To sp.011 AOX mitochondria

respiring on NADH a saturating amount of ADP was added, inducing a state 3, during this

state a saturating amount of OG was added to inhibit AOX. If AOX had been competing

with the cytochrome pathway then upon inhibition, electron flow would have been diverted

from the alternative pathway to the cytochrome pathway. Because of the proportional

relationship between and vO2 under state 3 conditions and due to an increase of

195

electron transfer rate through the cytochrome pathway should increase. However,

addition of OG during state 3 had no effect on whatsoever, see Figure 5.12. This was

seen with either NADH or succinate as a substrate. This indicates that under state 3

conditions the alternative and cytochrome pathway do not compete for reducing equivalents

and there is no diversion of electron flow from the alternative pathway to the cytochrome

pathway upon inhibition of AOX. It suggests that the activities of both pathways are

additive.

5.3.5 Comparing sp.011 AOX and sp.011 AOX+T oxidising pathway kinetics with succinate

as a substrate—The oxidising pathway kinetics of sp.011 AOX mitochondria respiring on

NADH (see section 5.3.3) were different from the kinetics obtained by Affourtit in a

previous study [93]. The oxidising pathway kinetics in that study however were determined

in sp.011 AOX and sp.011 AOX+T mitochondria respiring on succinate. The differences

between the kinetics obtained in this study and Affourtit’s study could be substrate

dependent. Therefore the oxidising pathway kinetics under various energetic conditions in

sp.011 AOX and sp.011 AOX+T mitochondria were determined with succinate as a

substrate. It was found, in the presence of AOX, under all energetic conditions, that at each

value of Qr/Qt the vO2 rates were higher when compared to sp.011 AOX+T mitochondria,

see figures 5.13-5.15. Similar to what was found with NADH in this study and opposite of

what was found by Affourtit. Under ADP limited conditions, the Qr/Qt vs. vO2 relationship

was linear, similar to what was seen in the wild type (see Figure 4.9). Under state 3 and

uncoupled conditions in sp.011 AOX+T mitochondria the relationship between Qr/Qt vs.

vO2 displayed the familiar biphasic pattern (see figures 5.14 and 5.15) as seen in the wild

type (see Figure 4.11). Under state 3 and uncoupled conditions in sp.011 AOX

mitochondria the relationship between Qr/Qt vs. vO2 is linear (see figures 5.14 and 5.15)

similar to what was seen in sp.011 AOX mitochondria respiring on NADH (see figures 5.8

and 5.9). From these results it was concluded that in the presence of AOX, under all

energetic conditions, at each value of Qr/Qt the vO2 rates are higher when compared to

sp.011 AOX+T mitochondria. Furthermore the differences between kinetics obtained in this

study and those of Affourtit’s study cannot be explained in terms of substrate dependent

differences.

196

5.3.6 Why are the oxidising pathway kinetics obtained in this study different from

Affourtit’s study?—The RCR value of sp.011 AOX+T mitochondria respiring on NADH

(uncoupled / state 2) in this study is about 5 (Table 5.3). The equivalent RCR value in

Affourtit’s study is estimated by this author to be around 2 (cf. Figure 2A in [93]). Also the

reaction medium used by Affourtit contains 0.3 M mannitol, whereas in yeast studies

normally a concentration around 0.65 M is used. To investigate the effect of the osmotic

strength of the reaction medium, state 3 oxidising pathway kinetics were determined for

sp.011 AOX mitochondria respiring on NADH in reaction medium containing 0.3 M

mannitol or reaction medium containing 0.65 M mannitol. Figure 5.16 shows that sp.011

AOX mitochondria in 0.3 M mannitol display lower vO2 rates than mitochondria respiring

in 0.65 M mannitol. Finally, in all experiments done by Affourtit ethanol was present in the

reaction medium (addition of Q1 dissolved in ethanol) which seriously affects oxidising

pathway kinetics by generating internal NADH and activating the internal NADH

dehydrogenase. Based on these differences it can be concluded that the kinetics reported in

[93] are completely incomparable to the kinetics determined in this study.

5.3.7 Are there any substrate dependent differences in sp.011 AOX oxidising pathway

kinetics?—It was found that under all energetic conditions with NADH as a substrate

higher maximum respiration rates were attained than with succinate as a substrate. This

could be due to dehydrogenase kinetics, with the external NADH dehydrogenase being

more active than SDH and reducing the Q-pool to a higher degree. However it can be seen

that under ADP limited conditions, from around 60% Qr/Qt at equivalent values of Qr/Qt

with NADH as a substrate the vO2 rates are higher than with succinate (see Figure 5.17).

Under state 3 and uncoupled conditions the same thing happens from around 40% Qr/Qt

(see figures 5.18 and 5.19). The Qr/Qt values at which point the oxidising pathway kinetics

with either NADH or succinate no longer overlap are estimated on the basis of the raw data

points. A more quantitative approach would be to compare mathematical fits through the

data; results from modelling the data, employing Q-pool kinetics (see section 2.4.5) will be

presented in chapter 6. At this point however it can be concluded that sp.011 AOX

mitochondria display substrate dependent differences in oxidising pathway kinetics.

197

5.3.8 Are the substrate dependent differences a characteristic of the cytochrome

pathway?—It was shown in chapter 4 that S. pombe mitochondria do not display any

substrate dependent differences in cytochrome pathway kinetics (see figures 4.9-4.12). It

can be argued however that transformation of S. pombe cells may affect mitochondrial

respiratory kinetics thus introducing substrate dependent differences in cytochrome

pathway kinetics which are not present in the wild type mitochondria. In order to

investigate this the cytochrome pathway kinetics of sp.011 AOX+T mitochondria respiring

on either succinate or NADH were plotted together, see figures 5.20-5.22. From these plots

it was concluded that there are no substrate dependent differences in the cytochrome

pathway kinetics of the transformed S. pombe mitochondria.

5.3.9 Are the substrate dependent differences a characteristic of the alternative

pathway?—Substrate dependent differences in alternative pathway activity are well known

[74, 254]. It is often found that alternative pathway respiratory rates with succinate are

higher than with NADH [72] , this being due to pyruvate formation during succinate

dependent respiration, pyruvate being an activator of plant AOX [79, 255]. When

mitochondria, expressing AOX, respiring on NADH are incubated with pyruvate, the

respiration rates attained are equivalent to what is seen with succinate. In our system

however AOX is constitutively active [70] and a difference in AOX activity dependent on

substrate is therefore not to be expected. This was investigated by determining alternative

pathway activity in sp.011 AOX mitochondria incubated in the presence of antimycin A

respiring on either NADH or succinate, see Figure 5.23. The reduction state of the matrix

pyridine nucleotide pool has been reported to activate AOX by reduction of the regulatory

sulfhydryl-disulfide system [256]. Inhibition of the cytochrome pathway can lead to a

perturbation of the reduction state of the pyridine nucleotide pool, increases in the

NAD(P)H / NAD(P)+ ratio can lead to rapid activation of AOX [71]. Inhibition of the

cytochrome pathway therefore could lead to activation of AOX. However, as reported

previously, S. guttatum AOX expressed in S. pombe mitochondria is constitutively active

[70]. From the Western blots in Figure 5.1 it is also apparent that AOX is in its reduced

state in S. pombe mitochondria. An increase in the reduction level of the matrix pyridine

198

nucleotide pool could therefore not lead to change of AOX activation status. It was

concluded that there are no substrate dependent difference in alternative pathway kinetics.

5.3.10 Are the substrate dependent differences a characteristic of the expression system

used?—In order to investigate whether or not the substrate dependent differences in sp.011

AOX oxidising pathway kinetics are a characteristic of our expression system a comparison

was made with oxidising pathway kinetics in a plant species naturally expressing AOX, A.

maculatum. It was demonstrated that mitochondria isolated from A. maculatum do not

display substrate dependent differences in oxidising pathway kinetics, see Figure 5.24.

5.3.11 Can the alternative pathway compete with the cytochrome pathway in S. pombe

mitochondria expressing AOX?—Inhibitor titrations according to the method of Bahr and

Bonner [252] were employed to generate -plots, see figures 5.25 and 5.26. From these

results it is clear that electron flow can be diverted from the cytochrome pathway to the

alternative pathway and vice versa. This indicates that neither pathway is saturated and that

the results in this study agree well with what is found in plant mitochondria with fully

activated AOX.

5.3.12 Does expression of the alternative oxidase lead to a change in Q-pool behaviour?—

Incorporation of an extra protein into the relatively packed IMM could lead to a change in

Q-pool behaviour. Figures 5.27 and 5.28 show that sp.011 AOX mitochondria display Q-

pool behaviour. Equally also, SDH activity in sp.011 AOX mitochondria is dependent upon

energy status of the IMM comparable to sp.011 wt mitochondria (cf. Figure 4.13).

199

5.4 CONCLUSION

The sp.011 AOX and sp.011 AOX+T oxidising pathway kinetics found in this study are

different from what has been reported previously [93, 153]. The expression of an extra

terminal oxidase results in overall higher respiratory rates, which is what one would expect

[29]. Given the differences in experimental conditions it can be concluded that the results

from this study are incomparable to the results obtained by Affourtit. The substrate

dependent differences in sp.011 AOX mitochondria are not caused by cytochrome pathway

kinetics, nor by alternative pathway kinetics. The substrate dependent differences are only

seen when both pathways are active simultaneously. In order to be able to quantitatively

determine at which value or Qr/Qt the substrate dependent oxidising pathway kinetics start

to diverge it is necessary to apply mathematical curve fitting to the data. Results of

mathematical modelling of the data will be presented in the next chapter. In order to gain

more insight into the nature of the substrate dependent differences experiments will be

presented where both NADH and succinate are used with the same mitochondria.

200

Chapter 6

Modelling of oxidising pathway kinetics in Schizosaccharomyces

pombe mitochondria expressing the alternative oxidase

6.1 INTRODUCTION

Substrate dependent differences in oxidising pathway kinetics in sp.011 AOX mitochondria

were described in the previous chapter. It was determined that substrate dependent

differences were not a characteristic of either the cytochrome or the alternative pathway.

Substrate dependent differences in kinetics were only seen when both pathways were active

at the same time. The biphasic pattern in state 3 and uncoupling oxidising pathway kinetics

(Qr/Qt vs. vO2) is masked with either NADH or succinate as substrate in sp.011 AOX

mitochondria. A biphasic pattern can still clearly be seen under state 3 conditions in the

vs. vO2 relationship. In order to more accurately determine at which value of Qr/Qt the

oxidising pathway kinetics start to diverge the raw data was fitted using reversible

Michaelis-Menten kinetics according to [76] (see section 2.4.5 for a detailed description).

With NADH as a substrate the combined activities of cytochrome pathway and AOX

appear to be additive, whereas with succinate the total oxidizing pathway activity appears

to be less than the sum of cytochrome and AOX pathway activities combined, see Table

5.2. These findings are suggestive of a possible limiting effect of succinate on total

oxidizing pathway activity. To investigate whether or not succinate indeed had such an

inhibitory effect experiments were done in which oxidising pathway kinetics were

determined using both NADH and succinate to reduce the Q-pool.

201

6.1 RESULTS

6.1.1 Modelling of sp.011 AOX oxidising pathway kinetics—In section 5.2.4.1 data was

shown which suggest that the oxidising pathway kinetics in sp.011 AOX mitochondria

respiring on either succinate or NADH start to diverge at low values of Qr/Qt, see figures

5.17-5.19. It was estimated for ADP limited conditions that the NADH and succinate

oxidising pathway kinetics start to diverge at around 60% Qr/Qt, whereas under state 3 and

uncoupled conditions this occurs at around 40% Qr/Qt. In order to arrive at a more accurate

determination of the point where NADH and succinate kinetics start to diverge, the data

was modelled using a reversible Michaelis-Menten equation according to [76]. Figure 6.1

shows the result of curve fitting the sp.011 AOX oxidising pathway data under ADP

limited conditions with either NADH or succinate as a substrate.

Figure 6.1 sp.011 AOX oxidising pathway kinetics under ADP limited conditions with either NADH (black

circles) or succinate (red circles) as substrate. Data taken from Figure 5.17. Curve fitting according to [76].

As opposed to a Qr/Qt value of 60%, as was estimated from Figure 5.17 (see section

5.2.4.1), after curve fitting, it can be seen that NADH and succinate kinetics start to diverge

at around 30% Qr/Qt which was not readily apparent from the raw data.

0

50

100

150

200

250

0 0.2 0.4 0.6 0.8 1

vO

2 n

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l O

2 /

min

/ m

g p

rote

in

Qr/Qt

202

A more pronounced difference between succinate and NADH oxidising pathway kinetics

under state 3 conditions in sp.011 AOX mitochondria was apparent from the data (cf.

Figure 5.18). This is also reflected in the fitted curves through these data, see Figure 6.2.

Figure 6.2 sp.011 AOX oxidising pathway kinetics under state 3 conditions with either NADH (black circles)

or succinate (red circles) as substrate. Data taken from Figure 5.18. Curve fitting according to [76].

Curve fitting reveals that succinate and NADH oxidising pathway kinetics start to diverge

at a much lower Q-redox poise than the previously estimated 40%. Based on the kinetic fits

it can be concluded that under state 3 conditions, at each value of Qr/Qt, with NADH as a

substrate the oxygen consumption rates are higher than with succinate as a substrate. Under

uncoupled conditions it appears that at low values of Qr/Qt, the oxygen consumption rates

with succinate as a substrate are slightly higher than with NADH, but at approximately

20% Qr/Qt the kinetics change considerably with oxygen consumption rates being higher at

each value of Qr/Qt with NADH as a substrate, see Figure 6.3.

0

50

100

150

200

250

300

350

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

rote

in

Qr/Qt

203

Figure 6.3 sp.011 AOX oxidising pathway kinetics under uncoupled conditions with either NADH (black

circles) or succinate (red circles) as substrate. Data taken from Figure 5.19. Curve fitting according to [76].

The data in figures 5.17-5.19 suggested the presence of substrate dependent differences in

oxidising pathway kinetics in sp.011 AOX mitochondria under various energetic

conditions. Modelling the data using reversible Michaelis-Menten kinetics (a method which

has been used successfully with isolated mitochondria in the past [21, 70, 76, 153, 216])

corroborates this. When the kinetic fits from figures 6.1-6.3 are combined in one plot, a

dependency on energy status is seen which was not readily apparent from the individual

figures, see Figure 6.4. The differences between succinate and NADH kinetics become

more pronounced when going from coupled to uncoupled conditions. Differences in

oxidising pathway kinetics should be independent from reducing pathway kinetics (see

section 1.5). Oxidising pathway kinetics reflect the relationship between steady state

respiration rate and steady state Qr/Qt. Differences in activity between dehydrogenases are

cancelled out when Q-pool kinetics [216] are assumed. In other words, the means with

which the Q-pool is reduced should not affect the oxidising pathway relationships. Still, it

was found that energy status affects SDH activity (cf. figures 3.19, 4.13 and 5.27) but not

that of the external NADH dehydrogenase (cf. Figure 4.15).

0

100

200

300

400

500

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

rote

in

Qr/Qt

204

Figure 6.4 Combined kinetic fits of sp.011 AOX oxidising pathway kinetics with NADH or succinate as

substrate. Each energetic state is indicated by colour (state 2 red, state 3 blue and uncoupled black). Of each

colour, the lower curve represents the succinate kinetics. Data taken from figures 6.1-6.3. N = NADH, S =

succinate.

Both SDH activity and the extent of substrate dependent differences in oxidising pathway

kinetics are dependent on energy status. Possibly these two effects are related, which could

challenge the model of Q-pool kinetics as it suggests interaction between reducing and

oxidising pathway activities in a non Q-dependent manner. It can be concluded that,

overall, oxidising pathway activity with succinate appears to be lower than with NADH as

a substrate. Possibly the total respiratory rate is lower than the sum of the individual

pathway activities with succinate as a substrate.

6.1.2 Are the oxidising pathway activities additive?—It was established that sp.011 AOX

mitochondria show distinct differences between oxidising pathway kinetics dependent on

substrate. Under all energetic conditions, over nearly the whole range of Qr/Qt, the oxygen

consumption rates with succinate are lower than with NADH as a substrate. Substrate

dependent differences are not seen in the cytochrome pathway kinetics (see section 5.3.7)

nor in the alternative pathway kinetics (see section 5.3.8).

0

100

200

300

400

500

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

rote

in

Qr/Qt

state 2

state 3

uncoupled

N

N

N

S

S

S

205

Substrate dependent differences are only apparent when the two pathways are active

simultaneously. Possibly with succinate as a substrate either one or both oxidising

pathways are engaged to a lower extent than with NADH as a substrate. Table 6.1 shows

the sp.011 AOX state 2 respiratory rates with either succinate or NADH as a substrate, data

taken from Table 5.2.

sp.011 AOX Respiratory Rate

(nmol O2 min-1

mg-1

protein)

substrate(s) state 2

NADH 199 (27)

NADH + OG 92 (10)

NADH + AA 81 (17)

succinate 142 (14)

succinate + OG 83 (12)

succinate + AA 84 (13)

Table 6.1 sp.011 AOX state 2 rates under various energetic conditions. Data taken from Table 5.2.

Assessing the engagement of oxidising pathways based on respiratory rates only, cannot be

used to quantify the contribution of each pathway to total respiration [148]. Respiration rate

data however can be used to give qualitative answers. The respiration rates given in Table

6.1 were obtained through addition of saturating amounts of substrate (NADH 1.8 mM or

succinate 9 mM). Therefore the level of Q-reduction is only limited by the activity of the

dehydrogenases. The steady state respiration under state 2 conditions with NADH is 199

nmol O2 / min /mg protein whereas with succinate it is 142 nmol O2 / min / mg protein. The

difference in rates could be due to a difference in reducing power of the dehydrogenases,

where the Q-pool is more reduced with NADH. It can be seen in Figure 6.1 that the Q-pool

with NADH is indeed more reduced (~10%). However, based on the curve fits through the

data, an increase in Qr/Qt to the same level as attained with NADH would increase the

206

respiration rate only marginally and cannot account for the discrepancy between oxygen

consumption rates in sp.011 AOX mitochondria respiring on either succinate or NADH

(saturating concentrations). The data in Table 6.1 were obtained from respiratory

measurements using the oxygen electrode only and therefore represent a dataset different

from the titration data presented in Figure 5.17. Respiratory rates obtained in both sets of

experiments are comparable which corroborates the hypothesis that there are substrate

dependent differences in sp.011 AOX oxidising pathway kinetics.

The activities of both pathways separately have also been determined, by incubating

with either octyl gallate or antimycin A. The cytochrome pathway respiratory rates for

NADH and succinate are 92 and 83 nmol O2 / min / mg protein respectively. The

alternative pathway respiratory rates for NADH and succinate are 81 and 84 nmol O2 / min

/ mg protein respectively26

. It appears that under ADP limited conditions, with NADH as a

substrate the activities of the cytochrome pathway and the alternative pathway are additive,

whereas with succinate as a substrate total respiration appears to be less than the sum of the

individual pathway activities. Under ADP limited conditions when only the cytochrome

pathway is active the Q-pool is reduced to the same extent with either succinate or NADH

as a substrate (cf. Figures 4.9 and 5.20) and respiratory rates are comparable.

Figure 5.23 suggests that with NADH as a substrate the Q-pool is more reduced than with

succinate as a substrate in sp.011 AOX mitochondria incubated in the presence of

antimycin A (only the alternative pathway active). With succinate as a substrate the Q-pool

was never reduced further than 80%. With NADH as a substrate in most cases the Q-pool

was not further reduced than 80%, except during two experimental days (out of five) where

the Q-pool was reduced somewhat more. It is tempting to conclude that there is a substrate

dependent difference in alternative pathway kinetics as it would offer a simple explanation

for the substrate dependent differences observed in total oxidising pathway kinetics,

without having to challenge Q-pool kinetics. However, the data do not allow for such a

generalisation since in the majority of traces, no substrate dependent differences in

alternative pathway kinetics were seen. If with NADH the Q-pool would be more reduced

than with succinate this would still only lead to an increase in AOX dependent respiration

26

Energy status has no effect on AOX activity and the respiratory rates are the same under all energetic

conditions.

207

of approximately 20 nmol O2 / min / mg protein (cf. Figure 5.23), which cannot account for

any of the discrepancies in oxygen consumption rates at specific Qr/Qt values with either

succinate or NADH. Any differences between NADH and succinate dependent alternative

pathway respiration cannot be due to limitation of SDH since under ADP limited conditions

both substrates can reduce the Q-pool to the same extent with only the cytochrome pathway

active. Under ADP limited conditions with both pathways active, it appears that with

NADH both pathways are fully active, whereas with succinate, even though the Q-pool is

reduced to almost the same extent, a much lower respiratory rate is reached, which suggests

that activity of either one or both pathways is/are limited in the presence of succinate.

To investigate whether or not the presence of succinate has a limiting effect on oxidising

pathway kinetics in sp.011 AOX mitochondria experiments were done where the Q-pool

was reduced with a mixture of substrates.

6.1.3 sp.011 AOX mixed titration studies—To establish a full reduction of the Q-pool and

maximum respiratory activity normally a combination of substrates are added to

mitochondria [74, 243, 257]. The results discussed in section 6.1.2 suggest that the presence

of succinate may have a limiting effect on sp.011 AOX respiratory kinetics when both

pathways are active. If this is the case then such an effect should be apparent when

oxidising pathway kinetics are determined using mixed substrate titrations of NADH and

succinate. The Q-pool can be partially reduced by titration with sub-saturating amounts of

NADH, in the presence of the NADH regenerating system, subsequently it can be further

reduced by addition of succinate and either ATP, glutamate or both (to fully activate SDH).

If succinate does not have any limiting effects it can be expected that the oxidising pathway

kinetics determined by partially reducing the Q-pool with NADH and by subsequently

reducing it further with succinate are comparable to oxidising pathway kinetics with just

NADH as a substrate. If succinate indeed has a limiting effect then the oxidising pathway

kinetics should be different from those obtained with NADH, in fact, at comparable levels

of Qr/Qt lower oxygen consumption rates should be obtained.

Apart from investigating a possible limiting effect of succinate on oxidising

pathway activities, the mixed substrate titrations serve as a control experiment to confirm

208

that there are indeed substrate dependent differences in sp.011 AOX oxidising pathway

kinetics. The data points in figures 6.1-6.3 suggest that succinate oxidising pathway

kinetics are different from NADH kinetics. Modelling of the data corroborates this.

However as can be seen in Figure 6.3, under uncoupled conditions SDH activity is limiting

as there are no succinate data points at Qr/Qt values of 50% and higher. Combining

substrates allows for reducing the Q-pool to higher Qr/Qt values and investigation of

succinate dependent kinetics at Q-reduction levels higher than 50%.

Substrate dependent differences between sp.011 AOX mitochondria are most

pronounced under uncoupled conditions (cf. Figure 6.4). Incubated in the presence of

CCCP (2 µM) sp.011 AOX oxidising pathway kinetics were determined using a mixture of

substrates. Figure 6.5 shows a representative trace of a 'mixed titrations' experiment. In the

presence of the NADH regenerating system sp.011 AOX mitochondria are titrated with

sub-saturating amounts of NADH in order to partially reduce the Q-pool, subsequently

succinate is added followed by ATP and/or glutamate. It can be seen that upon addition of

G-6-P both Qr/Qt and vO2 increase (endogenous NAD+ is converted to NADH).

Subsequent addition of NADH (~2.5 M) leads to a proportional increase in activity.

Further addition of NADH (~5 M) leads to a non-proportional increase in activity

associated with the biphasic point (the biphasic pattern is masked but still present in sp.011

AOX mitochondria, cf. Figure 5.9). The inset shows what the oxidising pathway kinetics

would look like if the NADH titration was continued (NADH titration done on the same

day with the same mitochondrial preparation), after the biphasic point, an increase of ~40%

Q-reduction yields a concomitant increase in vO2 of about 400 nmol O2 / min / mg protein

with NADH. However, when succinate (9 mM) and ATP (0.2 mM) are added to further

reduce the Q-pool (after partial reduction with NADH) the increase in vO2 is only about 40

nmol O2 / min / mg protein, whereas Qr/Qt is increased by about 30%. Activity is

effectively limited by the addition of succinate and further activation of SDH.

Figure 6.6 shows a similar titration where the Q-pool was further reduced (~50%)

initially with sub-saturating amounts of NADH before succinate was added. Beyond the

biphasic point the relationship between Qr/Qt and vO2 becomes very steep, addition of a

third aliquot of NADH results in an increase in Qr/Qt of ~15% with a concomitant increase

in vO2 of about 140 nmol O2 / min / mg protein. Subsequent addition of succinate (9 mM)

209

leads to a ~25% increase in Qr/Qt whereas vO2 hardly changes, which indicates that with

succinate as a substrate the combined oxidising pathway activities become limited.

Figure 6.5 A representative trace of a mixed substrate titration under uncoupled conditions in sp.011 AOX

mitochondria. Glucose-6-phosphate dehydrogenase is incubated. Titration is started with addition of glucose-

6-phosphate (G6P) followed by two additions of subsaturating amounts of NADH (~7 M) leading to a

partially reduced Q-pool (40%). Subsequently succinate (9 mM) and ATP (0.2 mM) are added which lead to a

further reduction of the Q-pool. The inset shows a representative trace of an NADH titration (0 – 75 M)

under uncoupled conditions in sp.011 AOX mitochondria. Mitochondrial protein used per experiment is 0.5-

0.7 mg.

0

50

100

150

200

250

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

ro

tein

Qr/Qt

G6P NADHNADH succinate ATP

0

100

200

300

400

500

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

ro

tein

Qr/Qt

210

Figure 6.6 A representative trace of a mixed substrate titration under uncoupled conditions in sp.011 AOX

mitochondria. Glucose-6-phosphate dehydrogenase is incubated. Titration is started with addition of glucose-

6-phosphate (G6P) followed by three additions of subsaturating amounts of NADH (~27 M) leading to a

partially reduced Q-pool (~50%). Subsequently succinate (9 mM) is added which leads to a further reduction

of the Q-pool (~75%). Mitochondrial protein used 0.5 mg.

Several things can be learned from figures 6.5 and 6.6. If there were no substrate dependent

differences in sp.011 AOX respiratory kinetics, then it should not matter in which way the

Q-pool is reduced. Clearly that is not the case, the kinetics displayed in the inset of Figure

6.5 with just NADH as a substrate look very different from the kinetics obtained when both

succinate and NADH are present. This experiment corroborates the hypothesis that

oxidising pathway kinetics in sp.011 AOX mitochondria are substrate dependent.

Addition of succinate (and subsequent addition of activators of SDH) limit NADH

dependent respiration. Upon addition of succinate (in the presence of a sub-saturating

concentration of NADH) the Q-pool is significantly reduced , the concomitant increase in

vO2 however is negligible when compared to NADH kinetics.

0

50

100

150

200

250

300

350

400

0 0.2 0.4 0.6 0.8 1

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2 /

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g p

rote

in

Qr/Qt

G6P NADHNADH succinateNADH

211

0

100

200

300

400

500

0 0.2 0.4 0.6 0.8 1

NADHmixed titrationssuccinate

vO

2 n

mo

l O

2 /

min

/ m

g p

rote

in

Qr/Qt

Figure 6.7 shows combined uncoupled sp.011 AOX oxidising pathway kinetics with

NADH, succinate and mixed substrates. From the mixed substrate data, the initial NADH

points are omitted, i.e. only the succinate and subsequent ATP and/or glutamate data points

obtained (by addition of these chemicals after the Q-pool was partially reduced with sub-

saturating amounts of NADH) are shown. The mixed substrate kinetics look different from

the NADH kinetics. Figure 6.8 shows the omitted NADH data from the mixed titrations

plotted in combination with uncoupled NADH oxidising pathway data. These data sets

overlap and confirm that the sp.011 AOX mitochondria used for the mixed titration studies

displayed regular NADH oxidising pathway kinetics.

Figure 6.7 Combined oxidising pathway kinetics in sp.011 AOX mitochondria respiring either on NADH,

succinate or both. NADH ( ) data points are taken from Figure 5.9. Succinate () data points are taken from

Figure 5.15. The mixed titration () data were obtained from four mitochondrial isolations, combining data

points from eight traces. The G6P and NADH data points from the mixed titration data are omitted in the

figure. Mitochondrial protein used per experiment is 0.5-0.7 mg.

212

0

100

200

300

400

500

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

rote

in

Qr/Qt

Figure 6.8 Uncoupled sp.011 AOX NADH oxidising pathway data points () combined with the omitted

NADH () data points from the mixed substrate titrations. Mitochondrial protein used per experiment is 0.5-

0.7 mg

It is clear that the mixed titration points look different from both the succinate and the

NADH data. To investigate whether the mixed substrate kinetics are similar to either

NADH or succinate oxidising pathway kinetics the data were modelled according to [76].

6.1.4 Applying Q-pool kinetics to fit sp.011 AOX mixed substrate oxidising pathway data—

Figure 6.9 shows kinetic fits of the uncoupled sp.011 AOX NADH and mixed titration data.

It can be seen that the oxidising pathway kinetics start to diverge at around 40% Qr/Qt.

Also the shape of both types of kinetics is different, whereas the NADH kinetics are

slightly concave at high Qr/Qt, the mixed substrate kinetic are clearly convex. At low

values of Qr/Qt, respiration with succinate appears to be higher than with NADH. These

characteristics are reminiscent of the uncoupled succinate kinetics (cf. Figure 6.3).

213

0

100

200

300

400

500

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

ro

tein

Qr/Qt

Figure 6.9 Combined uncoupled oxidising pathway kinetics in sp.011 AOX mitochondria respiring either on

NADH or mixed substrates. NADH () data points are taken from Figure 5.9. The mixed titration () data

was taken from Figure 6.6. Using Q-pool kinetics the data were fitted. It is clear that the modelled kinetic

curves for both sets of data are different from one another. Curve fitting according to [76].

Figure 6.10 shows kinetic fits of the uncoupled sp.011 succinate and mixed titration data.

The oxidising pathway kinetics start to diverge at around 15% Qr/Qt. The shape of both

curves are clearly convex.

The mixed substrate oxidising pathway kinetics cannot be fitted exclusively by either

NADH or succinate kinetic fits. This suggests that both dehydrogenases are active.

The similarity in shape of the succinate and the mixed substrate oxidising pathway kinetics

suggest that SDH has a stronger control on oxidising pathway activity than the external

NADH dehydrogenase. This will be discussed in terms of reducing pathway kinetics in the

next section.

214

Figure 6.10 Combined uncoupled oxidising pathway kinetics in sp.011 AOX mitochondria respiring either on

succinate or mixed substrates. Succinate () data points are taken from Figure 5.15. The mixed titration ()

data was taken from Figure 6.6 Using Q-pool kinetics the data were fitted. It is clear that the modelled kinetic

curves for both sets of data are different from one another. Curve fitting according to [76].

0

50

100

150

200

250

300

350

400

0 0.2 0.4 0.6 0.8 1

vO

2 n

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l O

2 /

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/ m

g p

ro

tein

Qr/Qt

215

6.3 DISCUSSION

In this study substrate dependent differences in sp.011 AOX oxidising pathway kinetics

were further explored. Oxidising pathway data were modelled according to [76] (figures

6.1-6.3) and it was determined that succinate and NADH dependent oxidising pathway

kinetics under ADP limited conditions start to diverge at a Q-reduction level of ~30%.

Under state 3 conditions, over the whole range of Qr/Qt respiratory activity with NADH is

higher than with succinate. Under uncoupled conditions the succinate and NADH

dependent oxidising pathway kinetics start to diverge at a Q-reduction level of ~20%.

These determinations would not have been possible on the basis of looking at the raw data

alone (cf. figures 5.17-5.19). Another characteristic of the substrate dependent differences

in sp.011 AOX oxidising pathway kinetics becomes apparent when kinetic curves obtained

under various energetic conditions are plotted together (Figure 6.4). The substrate

dependent differences in kinetics become more pronounced upon deenergization of the

IMM.

Experiments done with the oxygen electrode showed that under ADP limited conditions the

respiratory rates obtained in the presence of NADH or succinate are different (Table 6.1).

This could be explained in terms of the ability of the dehydrogenases to reduce the Q-pool

with the external NADH dehydrogenase having more capacity than SDH. The Q-pool is

slightly more reduced with NADH than with succinate (cf. 6.1), but this argument would

only be relevant if both succinate and NADH data were on the same kinetic curve, which

they are not. Suppose there was in fact only one kinetic curve, either the succinate or the

NADH one in Figure 6.1, a ~10% increase in Qr/Qt would not lead to an increase in vO2

that could account for the differences in steady state respiratory rates with either succinate

or NADH as a substrate. The combined activities of the cytochrome pathway (determined

in the presence of OG) and the alternative pathway (determined in the presence of

antimycin A) with NADH as a substrate appear to be additive. With succinate as a substrate

the total respiratory activity appears to be less than the sum of the activities of the two

pathways. This suggests that succinate has a limiting effect on oxidising pathway kinetics.

It was already established that succinate has no limiting effects on either the cytochrome or

216

the alternative pathway as no substrate dependent differences are seen in either cytochrome

pathway kinetics (cf. figures 4.9,4.11,4.12 and 5.20-5.22) or alternative pathway kinetics

(cf. Figure 5.23). Also, it was determined in previous work done in this laboratory that

addition of succinate to sp.011 AOX mitochondria respiring on NADH does not have any

effect on respiration (cf. Table 6.1 in [26]), i.e. succinate does not have an inhibitory effect

on AOX per se. Therefore, when both pathways are active, one or both of them are limited

by succinate dependent respiration. A series of experiments were conducted where the Q-

pool was reduced with a mixture of NADH and succinate. This approach was taken to

address three interlinked questions:

1) Experiments using the oxygen electrode and titration studies using a combination of the

Q-electrode and the oxygen electrode revealed substrate dependent differences in oxidising

pathway kinetics, this was further corroborated by modelling the data. To confirm the

presence of these substrate dependent differences oxidising pathway kinetics in the

presence of both NADH and succinate were determined.

2) To investigate any limiting effects of succinate on oxidising pathway kinetics succinate

(and SDH activators) were added to sp.011 AOX mitochondria respiring on NADH.

3) According to Q-pool kinetics the oxidising pathway kinetics are only dependent on the Q

redox poise, regardless as to how the Q-pool is reduced. Reducing the Q-pool with both

succinate and NADH will reveal whether in sp.011 AOX mitochondria Q-pool kinetics

apply.

Addition of succinate (and SDH activators) to sp.011 AOX mitochondria respiring on sub-

saturating amounts of NADH (in the presence of the NADH regenerating system) leads to a

large increase in Qr/Qt with a concomitant low increase in vO2 leading to oxidising

pathway kinetics which are unlike the kinetics seen with just NADH (figures 6.5 and 6.6).

These findings suggest that succinate indeed has a limiting effect on oxidising pathways

activity and it confirms that there are substrate dependent differences in sp.011 AOX

oxidising pathway kinetics. Furthermore, these results suggest that Q-pool kinetics may not

apply (discussed later). At Qr/Qt levels of 20% and higher the respiratory rates in the

presence of NADH and succinate are higher than with succinate alone (Figure 6.7). This

suggests that both dehydrogenases are active and that activity of SDH does not inhibit the

217

0

50

100

150

200

250

300

0 0.2 0.4 0.6 0.8 1

vO

2 n

mo

l O

2 /

min

/ m

g p

rote

in

Qr/Qt

external NADH dehydrogenase. Curve fitting shows that the mixed substrate oxidising

pathway kinetics resemble succinate kinetics more closely than NADH kinetics (figures 6.9

and 6.10). These results suggest that in the presence of both NADH and succinate SDH has

more control on respiration than the external NADH dehydrogenase. In order to investigate

why the mixed substrate kinetics are more ‘succinate-like’ than ‘NADH-like’ it is necessary

to compare the reducing pathway kinetics. Figure 6.11 shows reducing pathway kinetics in

S. pombe mitochondria for both SDH and the external NADH dehydrogenase. It can be

seen that the relationship between Q-redox poise and oxygen consumption rate with the

external NADH dehydrogenase in S. pombe mitochondria is very steep, i.e. a small change

in Qr/Qt will result in a large change in vO2. These kinetics agree well with previously

published results on external NADH dehydrogenase kinetics in potato mitochondria (cf.

Figure 5 in [76]). The SDH kinetics display a considerably lower slope than the external

NADH dehydrogenase kinetics, i.e. a small change in Qr/Qt will result in a relatively small

change in vO2.

Figure 6.11 Reducing pathway kinetics in S. pombe mitochondria. SDH data points () are taken from

Figure 4.13. The external NADH dehydrogenase data points () are taken from Figure 4.3B in [26]. The

comparison is justified as the S .pombe mitochondria isolated in that study are essentially the same as in this

study and the results were obtained during the same time when results for this study were obtained, i.e. both

researchers were working on the same system in the same period. Concentrations: succinate 9 mM (SDH

activated with ATP (0.2mM) and glutamate (9 mM)), NADH 1.8 mM. Kinetics are modelled according to

[76].

218

The difference in slopes and shapes of the kinetic curves in Figure 6.11 reflect a difference

in affinity for ubiquinone (the substrate for the dehydrogenases), see Figure 6.12. The

reversible Michaelis-Menten equations used to model Q-pool kinetics can be condensed

into a simple equation:

(equation 2.6, section 2.4.5)

The parameter represents the affinity for ubiquinone and determines the shape (and

therefore the slope) of the kinetic curves. A low value for (e.g. –0.99) represents a high

affinity for ubiquinone, whereas a high value represents a low affinity, i.e. the shape of the

kinetic fits reveal at a glance any differences in substrate affinity. It is clear from Figure

6.11 that over the whole range of Qr/Qt SDH has a higher affinity for ubiquinone, which

means that at increasing levels of Q-reduction (decreasing concentration of ubiquinone)

SDH could ‘out-compete’ the external NADH dehydrogenase.

The oxidising pathway kinetics of sp.011 AOX mitochondria respiring on either NADH or

succinate look distinctly different. Mixed titration oxidising pathway kinetics do not

overlap with either type of kinetics, but look more similar to succinate kinetics than NADH

kinetics. A difference in affinity for ubiquinone may explain why SDH appears to have

more control over oxidising pathway activity. However a difference in dehydrogenase

kinetics cannot explain the occurrence of substrate dependent differences in oxidising

pathways since S. pombe mitochondria expressing only one pathway do not display any

substrate dependent differences.

v s

s 1

219

Figure 6.12 Reducing pathway kinetics modelling different affinities for ubiquinone. Curve 1 represents

modelled reducing pathway kinetics with a low value of , this represents a typical SDH kinetic curve. Curve

2 represents modelled reducing pathway kinetics with a high value of , this represents a typical external

NADH dehydrogenase curve. Respiratory rates in arbitrary units, figure adapted from Figure 3 in [216].

In order for dehydrogenases to have an effect on oxidising pathway kinetics a deviation

from Q-pool kinetics has to be assumed. There are several not mutually exclusive

possibilities:

1) The Q-pool in S. pombe mitochondria expressing both the cytochrome and the

alternative pathways is not homogeneous.

2) Either one or both of the dehydrogenases can interact with the oxidising pathways in a

non Q-dependent manner.

0

5

10

15

20

0 0.2 0.4 0.6 0.8 1

res

pir

ati

on

ra

te A

U

Qr/Qt

1

2

220

It has been shown that the Q-pool in S. pombe mitochondria displays pool behaviour (cf.

figures 4.13, 4.14, 5.27 and 5.28 and [26]). That would mean that with the introduction of

an extra oxidising pathway the Q-pool should become non homogenous. The introduction

of AOX itself does not lead to a deviation from pool behaviour (cf. Figure 5.27). Also in the

presence of antimycin A (only the alternative pathway active) no substrate dependent

differences are seen (cf. Figure 5.23). Therefore a deviation from pool behaviour would

occur when both pathways are active. Maybe, by introducing an extra oxidase the demand

for reducing equivalents exceeds the ability of SDH to keep the Q-pool homogeneously

reduced, i.e. there would be regional differences in QH2 concentration so that the bc1

complex and AOX would face different concentrations of substrate. The strain on SDH to

keep the Q-pool homogeneously reduced would increase if either the demand for reducing

equivalents by the oxidising pathways increased or if SDH itself would become

deactivated. Both happen during deenergization. Under state 3 or uncoupled conditions the

protonmotive force decreases and the cytochrome pathway is no longer restrained by it,

Figure 6.4 shows that (with either succinate or NADH as a substrate) upon going from

coupled (ADP limited) conditions to uncoupled (presence of ADP or CCCP) conditions the

oxidising pathway activity increases. Therefore upon increased deenergization the demand

for reducing equivalents from the Q-pool increases.

It was shown that SDH activity was dependent on energy status (cf. figures 3.19, 4.13 and

5.27). Therefore upon increased deenergization the ability of SDH to supply reducing

equivalents to the Q-pool decreases and the Q-pool becomes functionally

compartmentalized. It is clear from Figure 6.4 that upon increased deenergization the

substrate differences become more pronounced. The external NADH dehydrogenase is not

affected by energy status and has more reducing capacity than the SDH therefore it is

expected that the external NADH dehydrogenase is able to keep the Q-pool homogenously

reduced in the presence of two oxidases. Perhaps the deviation from Q-pool behaviour is

due to a physical limitation of the expression system where SDH is not able to keep the Q-

pool homogenously reduced. Deviation in Q-pool kinetics was not seen in a system

naturally expressing AOX (cf. Figure 5.24) suggesting that under natural conditions SDH is

able to keep the Q-pool homogenously reduced. The extra activity induced by functionally

expressing AOX in S. pombe mitochondria is not trivial, under ADP limited conditions

221

with NADH as a substrate the oxygen consumption rate increases by about 100 nmol O2 /

min / mg protein, double the rate with only the cytochrome pathway active, incorporation

of AOX leads to a considerable increase in demand for reducing equivalents from the Q-

pool. Perhaps the ETC in S. pombe mitochondria, as there never was any evolutionary

pressure to accommodate an extra oxidase, has no means of upregulating SDH expression

in response to an increased demand for reducing equivalents.

Another possibility could be that reducing pathway activities influence oxidising pathway

activities in a non Q-dependent manner. To explain an interplay found between cytochrome

and alternative pathway activity in mitochondria isolated from the amoeba Acanthamoeba

castellanii Jarmuszkiewicz et al. [258] postulate an effect of matrix pH on AOX activity.

An effect of pH on cyanide-resistant respiration has been found in mitochondria isolated

from A. castellanii [82] and from several plant sources such as vita bean (Vigna

unguiculata L.) [259], Arum italicum [260] and (important to this study) S. guttatum [83].

Due to cytochrome pathway proton pump activity the matrix pH can be changed. In the

presence of antimycin A, matrix pH would not change and no substrate dependent

differences would be seen in alternative pathway activity (cf. Figure 5.23). In the presence

of octyl gallate, or in S. pombe mitochondria not expressing AOX a difference in matrix pH

can be expected as cytochrome pathway activity with succinate is lower than with NADH

as succinate dependent respiration becomes limiting under state 3 and uncoupled

conditions, see figures 4.11 and 4.12. So possibly, when both pathways are active, with

NADH, a higher rate of proton pumping is achieved than with succinate, leading to

different matricial pH values depending on substrate, which affects AOX activity.

In section 4.2.2.1 it was discussed that nigericin (which abolishes pH [1]) had no effect on

which suggests that no pH was present (due to K+/H

+) activity. A pH effect on AOX

in mitochondria lacking an apparent pH would therefore not be expected, however it was

shown in V. unguiculata mitochondria that AOX was affected by pH even when nigericin

was present in the medium [259]. A mechanism like this would be compatible with the

results presented in this study. When the cytochrome pathway is active matrix pH values

could be different depending on substrate. Cytochrome pathway activity is not affected by

pH (or not to the same extent as AOX) and no substrate dependent differences would be

222

seen in cytochrome pathway kinetics. In the presence of antimycin A the cytochrome

pathway would be inactive and the pH would be the same with either succinate or NADH

and no substrate dependent differences would be seen in alternative pathway kinetics.

When both pathways are active, with the cytochrome pathway more active with NADH as a

substrate than with succinate there could be a substrate dependent difference in matrix pH

which specifically affects AOX activity, which would only be apparent when both

pathways are active.

6.4 CONCLUSION

The substrate dependent differences in sp.011 AOX oxidising pathway kinetics are only

seen when both oxidising pathways are active. In this study an alternative oxidase from a

thermogenic species (S. guttatum) was functionally expressed in S. pombe. Substrate

dependent differences were not found in another thermogenic species (A. maculatum),

naturally expressing AOX. One way to interpret this would be to assume that the substrate

dependent differences are a specific characteristic of our expression system.

Introduction of an extra oxidase to the ETC of S. pombe mitochondria might put a strain on

the system inducing deviation from Q-pool kinetics. In our laboratory S. pombe has also

been used to express the Arabidopsis thaliana alternative oxidase [70]. It would be of

interest to determine whether or not this system also displays substrate dependent

differences. Other possible avenues to explore would be to inhibit AOX27

in sp.011 AOX

mitochondria and determine if the substrate dependent differences disappear. In the event

of the substrate dependent differences being a characteristic of the expression system and

not a reflection of an in vivo situation our expression system is still very useful to address

different questions on a more fundamental level concerning Q-pool kinetics, the interplay

between pathways and to determine the limitations of dehydrogenases to keep the Q-pool

homogeneously reduced.

27

Difficult as the Q-electrode reacts strongly with octyl gallate, requiring long incubation times with OG for

the Q-signal to stabilise.

223

Chapter 7

General Discussion

In this study the yeast Schizosaccharomyces pombe has been used to functionally express

the plant Sauromatum guttatum alternative oxidase in order to investigate the influence of

AOX on respiratory kinetics. The alternative oxidase is non-protonmotive [261], i.e. the

free energy released upon oxidation of ubiquinol is not stored in an electrochemical

gradient of protons across the IMM. The activity of AOX leads to the dissipation of free

energy. Another means for many mitochondria to lower the efficiency of energy

transduction is via the uncoupling protein [262] which catalyses the transport of protons

into the matrix thereby decreasing the magnitude of the protonmotive force. It is unclear

whether or not AOX activity can lead to a decrease in the magnitude of p. It was

determined previously that uncoupling had no effect on AOX activity [176]. In this study

AOX activity was also found to be independent of energy status. S. pombe mitochondria do

not express complex I [13] therefore p is generated through the combined proton pumping

activities of complexes III and IV (see Figure 1.14). It has been found that in the presence

of pyruvate the alternative pathway can compete with the cytochrome pathway for reducing

equivalents [79]. Addition of pyruvate to S. pombe mitochondria expressing S. guttatum

AOX does not lead to an increase in activity, it was concluded that the alternative oxidase

in our expression system is constitutively active [70]. Since S. guttatum AOX in S. pombe

mitochondria is fully activated it can be hypothesized that the alternative pathway is able to

compete with the cytochrome pathway for reducing equivalents in a way similar to the case

in soybean mitochondria [79]. If indeed reducing equivalents were diverted from the

cytochrome pathway to the alternative pathway this might affect the membrane potential. In

order to investigate this a TPP+-electrode [154] was employed to determine the membrane

potential. The respiratory characteristics of the wild type S. pombe mitochondria had been

characterised to a certain extent previously [26, 104, 105]. The membrane potential

however (to the best of our knowledge) had not been determined previously. Prior to

224

investigating any effects of AOX activity on in S. pombe it was necessary to get an idea

of the bioenergetics in the wild type mitochondria. Also, due to several small alterations to

the isolation method (omitting the final PercollTM

purification step, using a bench top

centrifuge for slow spins instead of a floor centrifuge) led to a higher yield and a small

increase in quality. For these reasons it was decided to recharacterise the S. pombe

mitochondria in this study which yielded some very unexpected respiratory kinetics.

7.1 Characterisation of the wild type S. pombe mitochondria

The sp.011 wt mitochondria displayed respiratory characteristics similar to mitochondria

from other sources (see figures 4.1 to 4.3 and Table 4.1). Membrane potential values were

similar to what was seen in other yeast mitochondria. Under ADP limited conditions the

familiar non-ohmic relationship between and respiratory rate was observed (see Figure

4.5 and cf. Figure 3.4). The relationship between Qr/Qt and oxygen consumption rate under

ADP limited conditions when the cytochrome pathway kinetics were determined using an

NADH regenerating system [146] was linear, similar to what is seen in mitochondria from

other sources (see Figure 4.4). However, under state 3 and uncoupled conditions, the

cytochrome pathway kinetics (Qr/Qt vs. vO2) showed a distinct biphasic pattern (see

figures 4.6 and 4.8). Especially interesting were the results obtained when determining the

vs. vO2 cytochrome pathway kinetics under state 3 conditions (see Figure 4.7). A

biphasic pattern can also clearly be seen in the relationship between membrane potential

and oxygen consumption rate (see inset B of Figure 4.7). This observation provided the first

indication of the fact that the cause of the biphasic kinetics was located downstream from

the Q-pool (the membrane potential in S. pombe mitochondria is generated by the

cytochrome pathway). Equally also, and Qr/Qt are measured with different electrodes,

which argues against the possibility that the biphasic patterns were artifactual (the

possibility that the biphasic patterns are due to experimental artefacts is thoroughly

explored in section 4.3.2). The cytochrome pathway kinetics were determined using the

NADH regenerating system as it has the advantage over the malonate titration method

(with succinate as substrate) of obtaining more data points during one experimental trace.

To rule out the possibility that the biphasic patterns were NADH dependent, the

225

cytochrome pathway kinetics were again determined, this time with succinate as a substrate

(see figures 4.9-4.12). No substrate dependent differences were seen which corroborated

the notion that the cause for the biphasic patterns was not a characteristic of the

dehydrogenases. The most conclusive observation came with the determination of oxidising

pathway kinetics in S. pombe mitochondria expressing the alternative oxidase, see figures

5.8 and 5.9. If an extra terminal oxidase is present the biphasic pattern under state 3 and

uncoupled conditions (Qr/Qt vs. vO2) becomes masked. Under state 3 conditions in the

vs. vO2 oxidising pathway kinetics in sp.011 AOX mitochondria the biphasic pattern can

still be clearly seen and looks no different from S. pombe mitochondria not expressing

AOX (see figure 5.11), i.e. the biphasic kinetics are still there and are a characteristic of the

cytochrome pathway.

Contemporary work by Trumpower et al. [33] suggested that the yeast bc1 complex

displays kinetics which could explain our findings. In order to investigate this possibility

we employed a spectrophotometric technique where ferricyanide was used as an artificial

electron acceptor (see figures 4.16 to 4.18). The results showed that respiratory kinetics of

S. pombe mitochondria are different from potato mitochondria. Also a biphasic pattern

appears to be present in the S. pombe data and absent in the potato data. Our data suggests

that it is indeed bc1 complex kinetics underlying the biphasic patterns observed under state

3 and uncoupled conditions. Trumpower and colleagues worked on bc1 complexes isolated

from S. cerevisiae mitochondria. If indeed the biphasic pattern can be explained in terms of

yeast bc1 complex kinetics then oxidising pathway kinetics of S. cerevisiae mitochondria

should display a biphasic pattern; which is what we found, see Figure 4.19.

7.1.1 How does cytochrome bc1 complex activity lead to biphasic cytochrome pathway

kinetics in S. pombe mitochondria?—The cytochrome bc1 complex in eukaryotic organisms

is a homodimeric multi-subunit inner mitochondrial membrane protein complex [263]

which catalyses the protonmotive Q-cycle [31] (see also section 1.2.6). The Q-cycle

mechanism does not require the cytochrome bc1 complex to be dimeric and the functional

relevance of its dimeric structure is unclear [33]. Bernard Trumpower over the years has

postulated several mechanisms of cytochrome bc1 complex activity which could provide a

‘raison d'être for the dimeric structure of the enzyme’. Publications by Trumpower and co-

226

workers on the functional significance of the dimeric structure of the cytochrome bc1

complex have been appearing as far back as 1990 [264] and the most recent one came out

in June 2005 [235]. The first mechanism that was proposed (1990) has over the years

evolved considerably, one can discern two stages where this mechanism was changed. The

first two mechanisms appear highly relevant with respect to this study, the most recent

mechanism is difficult to reconcile with my results.

The early work In 1990 a publication came out in which half-of-the-sites reactivity of the

cytochrome bc1 complex in S. cerevisiae was reported [264]. In this study the nuclear gene

encoding for subunit 6 was deleted. This subunit is believed to be involved in binding

cytochrome c in cooperation with cytochrome c1. This deletion strain could be grown on

non-fermentable media indicating that the cells had a (partially) functional mitochondrial

respiratory chain. Isolated bc1 complexes from this strain showed a 50% reduction in

cytochrome c reductase activity when the salt concentration in the assay was similar to the

expected intracellular ionic strength. This reduced activity was not seen when the ionic

strength in the assay was lowered. The wild type bc1 complex activity did not show any

such dependency on ionic strength. It was further found in the deletion strain that under

conditions where half of the bc1 complexes are inactive all of the complexes retain the

ability to bind inhibitors (myxothiazol in this case). It was proposed that the genetic

deletion of subunit 6 mimics a reversible conformational change or dissociation of this

complex thereby inactivating one of the monomers in the dimeric complex. It was

hypothesized that a physiological effector which could interact with subunit 6 is the

protonmotive force. Conformation of subunit 6 might change upon a change in membrane

potential or a change in local pH. Under ADP limited conditions subunit 6 would be in a

conformation such that only one of the monomers in the bc1 complex is active.

If in S. pombe mitochondria the cytochrome bc1 complex behaves in a similar way as in S.

cerevisiae then a membrane potential induced conformational change in subunit 6 could

explain why the cytochrome pathway kinetics are biphasic under state 3 and uncoupled

conditions but not under ADP limited conditions.

227

Under state 2 and state 4 conditions, due to a high subunit 6 would be locked in a

conformation in which only one of the monomers per dimer is active. Upon decrease of

a conformational change occurs upon which both monomers now can become active

simultaneously. However, the fact that both monomers now can be active does not mean

that they will or that they would be equally active. This change in conformation does not in

itself explain the occurrence of a biphasic pattern unless the monomers within the dimer

become active sequentially. This is exactly what Trumpower et al. found next.

The intermediate work After a twelve year hiatus28

several publications on the yeast bc1

complex reaction mechanism came out [33, 34, 236]. It was found that several inhibitory

analogs of ubiquinol binding at center P could inhibit the wild-type S. cerevisiae enzyme

with a stoichiometry of 0.5 per bc1 complex. One molecule of inhibitor could fully inhibit

the dimeric enzyme. From this it was concluded that only one monomer per dimer was

active at a time, displaying the so called half-of-the-sites reaction mechanism. This was

found with stigmatellin and MOA-stilbene29

, but interestingly enough not with

myxothiazol. This is peculiar because both myxothiazol and MOA-stilbene are structurally

similar and have the same mechanism of inhibition (both inhibitors prevent electron flow

from ubiquinol to the iron-sulfur protein), it was suggested that very subtle differences in

ligand-protein interaction could account for this. Stigmatellin allows reduction of the iron-

sulfur protein but traps its position proximal to cytochrome b thereby preventing oxidation

by cytochrome c1. Inhibition with antimycin (which prevents reduction of ubiquinone or

ubisemiquinone at the N site) led to a stoichiometry of 1 per bc1 complex. Based on these

observations it was suggested that the yeast cytochrome bc1 complex oxidises ubiquinol by

a half-of-the-sites mechanism. Further inhibitor studies using stigmatellin or MOA –

stilbene showed that the yeast bc1 complex binds these inhibitors in an anti-cooperative

way. A second molecule of inhibitor binds with much lower affinity to a dimer in which an

inhibitor is already bound. Trumpower et al. propose that ubiquinol binding is also anti-

cooperative and that binding of ubiquinol to one monomer raises the Kd for ubiquinol

28

At the EBEC 2004 meeting in Pisa professor Trumpower, during a presentation, mentioned the fact that

the “half-of-the-sites reaction mechanism in the yeast bc1 complex” research line had been shelved

right after the 1990 publication. 29

MOA-stilbene – methoxyacrylate - stilbene

228

binding in the other monomer. Based on these findings the regulation of the bc1 complex in

yeast is hypothesized to occur in either of two ways:

1: Due to a conformational change the bc1 complex can switch from half-of-the sites

reactivity to full activation.

2: The bc1 complex functions with two affinities for substrate and the second half of

the dimer is only active at high ubiquinol concentration.

In the early work it was suggested that under ADP limited conditions subunit 6 is in a

conformation in which one of the monomers is inactivated. A physiological effector, such

as the protonmotive force, could reversibly change this conformation, leading to full

activation of the bc1 complex. Interestingly enough Trumpower does not link his earlier

suggestion to hypothesis 1. It was concluded that: ‘one can only speculate as to what types

of signals might bring about such a switch in the enzyme’. In S. pombe mitochondria the

biphasic pattern is seen only under state 3 or uncoupled conditions, i.e. a change in with

respect to ADP limited conditions. So possibly this change in leads to a conformational

change after which both monomers become active. This however does not explain the

occurrence of the biphasic pattern itself.

Hypothesis 2 could accommodate for this. The relationship between Qr/Qt vs. vO2 becomes

biphasic at around 40% Q-reduction under state 3 or uncoupled conditions in S. pombe

mitochondria. So perhaps at 40% Qr/Qt half of the high affinity monomers are bound and

upon a further increase in Q reduction, ubiquinol starts binding to the low affinity

monomers. However if the biphasic oxidising pathway kinetics are simply a reflection of

Qr/Qt reaching a threshold value why is there no biphasic pattern under state 2 or state 4

conditions? Under ADP limited conditions the Q-pool is further reduced than under state 3

or uncoupled conditions. Hypotheses 1 and 2 are not mutually exclusive. In order to explain

the biphasic kinetics in S. pombe mitochondria a mechanism which requires two

conformational changes is described next. Under ADP limited conditions only one

monomer is active, upon a change in a conformational change occurs and the second

monomer can now become active as well. Under state 2 or state 4 conditions this

229

conformational change never occurs and hence only one monomer per dimer is accessible

to ubiquinol. When one monomer in a dimer is bound the binding affinity in the other

monomer is increased. So even if both monomers become active upon a change in first

all high affinity monomers must be activated by ubiquinol before ubiquinol can bind to the

low affinity monomers. This could explain the occurrence of the biphasic pattern and why

it only occurs under conditions where is decreased.

The recent work Between 2004 and 2005 several papers came out [235, 265] which

focus on electron transfer between monomers in the bc1 complex via the bL cytochromes.

The distance between the bL heme in one monomer to the other bL heme in the second

monomer is 10-11 Å, this distance is bridged by three pairs of aromatic residues via which

electron transfer could take place30

. Interestingly enough Trumpower states that ‘Some

center P inhibitors have been shown to completely block bc1 complex activity upon binding

to only half of the dimeric complex, suggesting anti-cooperative interaction between the

ubiquinol oxidation sites in the dimer’ [265]. It is not readily apparent to this author how

half-of-the sites reactivity suggests anti-cooperative interaction. In the early work it was

established that under specific conditions the bc1 complex displayed half-of-the sites

reactivity, this was not related to anti-cooperative binding of ubiquinol. Only during the

intermediate work, from inhibitor studies it became apparent that the affinity for certain

center P inhibitors on the second monomer was decreased if another certain center P

inhibitor was already bound to the first monomer. This might explain the half-of-the sites

reactivity, not the other way around. It is suggested in the recent work that one active P site

can reduce two bH hemes (via intermonomer electron transfer). It is not clear how this

mechanism relates to anti-cooperative binding of ubiquinol. Concluding, the latest work by

Trumpower et al. on the regulation of the yeast bc1 complex does not seem to be a further

development of the previous mechanisms described in the early and intermediate work but

seems to be going on a tangent, no longer directly related to previous work nor related to

the results described in this thesis.

30

Theoretically it could also be possible for electron transfer to occur without participation of the

aromatic residues via electrontunneling [5].

230

7.1.2 Future work suggestions pertaining to the biphasic patterns in S. pombe cytochrome

pathway kinetics—The S. pombe spectroscopy data is somewhat limited. It can be seen in

Figure 4.18 that the data points are not equally distributed over the NADH concentration

range. It would be good if some extra experiments were done in which the whole

concentration range was covered.

It was hypothesized that the membrane potential was instrumental in the generation of the

biphasic pattern in the oxidising pathway kinetics. It would be interesting to see if in the

presence of sub-saturating concentrations of uncoupler, during the transition from coupled

to uncoupled conditions a biphasic pattern gradually becomes more apparent.

The S. cerevisiae data convincing as they may appear are from one experimental day only.

This experiment should be repeated.

Our data suggest that the biphasic patterns in oxidising pathway kinetics are a general

characteristic of yeast bc1 complex kinetics. It would be very interesting to see if isolated S.

pombe bc1 complexes display the same kinetics as their S. cerevisiae counterparts.

231

7.2 Functional expression of AOX in S. pombe mitochondria yields

substrate dependent differences in oxidising pathway kinetics.

Having characterised the wild type S. pombe mitochondria we subsequently started to

investigate the effects of heterologously expressed AOX in S. pombe mitochondria on

respiratory kinetics and the membrane potential. Previous work had shown that under

various energetic conditions the functional expression of the S. guttatum AOX in S. pombe

mitochondria led to a decreased oxygen consumption rate at a certain value of Qr/Qt when

compared to transformed S. pombe mitochondria in which AOX expression was repressed

[93]. This result is counter intuitive. One would expect that the introduction of an extra

terminal oxidase would lead to an overall increased oxygen consumption rate. This is

exactly what we found in this study, see figures 5.7 to 5.9. Using the NADH regenerating

system it was determined that under all energetic conditions (ADP limited, state 3 and

uncoupled) in the presence of AOX at a certain value of Qr/Qt the oxygen consumption rate

was higher when compared to S. pombe mitochondria in which AOX expression was

repressed. Possible causes for the differences between the results between this study and the

previous one done in this laboratory [93] were explored (see sections 5.2.3 and 5.3.6) and it

was concluded that it was a combination of differences in RCR, reaction medium used and

the fact that some of the substances added during Affourtit’s experiments were dissolved in

ethanol.

Apart from increased oxygen consumption rates it also became apparent that in the

relationship between Qr/Qt vs. vO2 under state 3 and uncoupled conditions the biphasic

pattern became masked (cf. figures 5.8 and 5.9). Whereas in transformed mitochondria in

which AOX expression was repressed the biphasic patterns were still to be seen (cf. figures

5.8 and 5.9). The biphasic pattern was still clearly present in the vs. vO2 relationship

under state 3 conditions in the sp.011 AOX mitochondria (which again corroborates the

notion that the biphasic pattern is a characteristic of the cytochrome pathway) and does not

look different from the same relationship in sp.011 AOX+T mitochondria. Importantly also

was the observation that under ADP limited conditions the familiar non-ohmic relationship

was observed between and vO2 in sp.011 AOX mitochondria, see Figure 5.10.

232

It can be seen that the relationship between vs. vO2 in sp.011 AOX mitochondria

overlaps with the sp.011 AOX+T data. This indicates that the introduction of the alternative

oxidase in the S. pombe IMM did not change its conductivity. It has been previously

hypothesized that the presence of protein complexes in the IMM leads to non-specific

proton leak [245]. The introduction of AOX to the S. pombe IMM might have affected its

conductive properties which would make comparisons between the respiratory kinetics of

mitochondria expressing AOX and mitochondria in which AOX is repressed problematic.

However, it is clear from Figure 5.10 that the IMM conductivities of both sp.011 AOX and

sp.011 AOX+T mitochondria are the same and any differences in respiratory kinetics

between the two types of mitochondria can be attributed to the activity of AOX.

7.2.1 Does AOX activity affect in S. pombe mitochondria ?—Under state 3 conditions

the relationship between and vO2 in the region after the inflection point of the biphasic

pattern is proportional, similar to what is seen in potato mitochondria (cf. inset A of Figure

4.7). This means that a decreased activity of the cytochrome pathway will lead to a

proportional decrease in . If in S. pombe mitochondria the alternative oxidase can

compete with the cytochrome pathway to such an extent that the cytochrome pathway

receives less reducing equivalents per unit time, then this should be reflected in a lowered

. It can be seen in Figure 5.11 that the titration data obtained with the NADH

regenerating system shows variation to such an extent that a possible effect of AOX activity

on cannot be discriminated. In a different approach to address this problem (see Figure

5.12) it became apparent that AOX activity does not affect the value of . This finding

suggested that AOX in S. pombe does not compete with the cytochrome pathway under

state 3 conditions.

7.2.2 Substrate dependent differences in oxidising pathway kinetics of S. pombe

mitochondria expressing AOX—The oxidising pathway kinetic data in S. pombe

mitochondria (described thus far) was obtained using the NADH regenerating system. The

results were different from a previous study [93] where oxidising pathway kinetics were

obtained using succinate as a substrate. To be certain, these experiments were repeated, see

figures 5.13 to 5.15. Again, it was observed that the introduction of an extra terminal

233

oxidase led to an increased oxygen consumption at a certain value of Qr/Qt when compared

to mitochondria in which AOX was repressed. Also, under state 3 and uncoupled

conditions the biphasic pattern in the relationship between Qr/Qt vs. vO2 is masked. The

oxidising pathway kinetics obtained with succinate as a substrate were different from the

results obtained in the previous study [93] and were in agreement with the NADH data, i.e.

with either succinate or NADH the biphasic pattern is masked and the oxygen consumption

rate at a certain value of Qr/Qt is higher in the presence of AOX than in the absence of

AOX.

Interestingly enough, when plotting the NADH and succinate data together it

became apparent that there were substrate dependent differences in oxidising pathway

kinetics, see figures 5.17 to 5.19. At a certain value of Qr/Qt the oxygen consumption rates

with NADH appear to be higher than with succinate. Under ADP limited conditions (Figure

5.17) the oxidising pathway kinetics seem to diverge at a Qr/Qt value of ~60% whereas

under state 3 and uncoupled conditions the kinetics seem to diverge at ~40%. These values

were estimated on the basis of the raw data. In order to get more accurate values the data

were fitted using a reversible Michaelis-Menten equation according to [76], see figures 6.1

to 6.3. Based on the kinetic fits it was estimated that under ADP limited condition the

kinetics start to diverge at a Qr/Qt value of ~30%. Under state 3 conditions oxygen

consumption rates with NADH as a substrate appear to be higher over the whole range of

Qr/Qt and under uncoupled conditions the kinetics start to diverge at a Qr/Qt value of about

20%.

7.2.3 What causes the substrate dependent differences in oxidising pathway kinetics in S.

pombe mitochondria expressing AOX?—Substrate dependent differences in total oxidising

pathway kinetics could be attributable to either of the individual pathways, the cytochrome

pathway or the alternative pathway. It was already shown in the wild type mitochondria

that the cytochrome pathway kinetics did not show any substrate dependent differences, see

figures 4.9 to 4.12. Possibly the act of transformation altered cytochrome pathway kinetics

leading to substrate dependent differences, this was also ruled out, see figures 5.20 to 5.22.

There were no substrate dependent differences in the cytochrome pathway kinetics of

sp.011 AOX+T mitochondria.

234

If not the cytochrome pathway, could there be substrate dependent differences in the

alternative pathway kinetics? This possibility was also ruled out, see Figure 5.23. No

substrate dependent differences were seen in sp.011 AOX mitochondria incubated in the

presence of AA. The substrate dependent differences are only seen when both pathways are

active simultaneously. As a comparison to a system in which AOX is naturally expressed

and where both pathways are active simultaneously we determined oxidising pathway

kinetics with either NADH or succinate as a substrate in mitochondria isolated from Arum

maculatum spadices, see Figure 5.24. No substrate dependent differences were seen. This

could be interpreted as such that the substrate dependent differences are a particularity of

our expression system.

7.2.4 Are the substrate dependent differences in oxidising pathway kinetics in S. pombe

mitochondria expressing AOX due to dehydrogenase characteristics?—According to the

Q-pool model (see section 1.5) the dehydrogenases do not interact directly with the

oxidising pathways, i.e. differences in reducing pathway activities are compensated for by

the Q-pool. The ubiquinol oxidases (bc1 complex and alternative oxidase) can only ‘sense’

the Q-redox poise. How this redox poise is brought about (through SDH or the external

NADH dehydrogenase) should not matter. However, upon combining the kinetic fits from

figures 6.1 to 6.3 in a composite figure (see Figure 6.4) it became apparent that the

substrate dependent differences in oxidising pathway kinetics became more pronounced

depending on the degree of deenergization of the IMM. It has been shown in both potato

and S. pombe mitochondria that SDH activity is dependent on energetic status31

, see figures

3.19, 4.13 and 5.27, whereas external NADH dehydrogenase activity is not dependent on

energy status, see Figure 4.15.

Possibly, in our expression system, with both pathways active, dehydrogenase activity

could determine oxidising pathway kinetics.

31

Which is probably not a direct effect on SDH but on succinate transport, see section 3.3.

235

It was shown that succinate appears to have a stronger control on oxidising pathway

kinetics, see figures 6.5-6.7, 6.9 and 6.10. When the Q-pool is partially reduced with sub-

saturating amounts of NADH, subsequent further reduction by addition of succinate (and

SDH activators) leads to kinetic curves which are more similar to the oxidising pathway

kinetics obtained with succinate than with NADH. In order to explain the substrate

dependent differences in terms of non Q-pool behaviour three observations are relevant:

1) SDH has a higher affinity for ubiquinone than the external NADH dehydrogenase (see

Figure 6.12).

2) SDH is physically located closer to the alternative oxidase (as both are facing the matrix)

than the external NADH dehydrogenase. Equally also the external NADH dehydrogenase is

physically located closer to the bc1 complex than SDH.

3) It has been shown that the Km of the alternative oxidase for duroquinol (3.3 mM) is

considerably higher than the Km for duroquinol of the cytochrome pathway [266], i.e. the

cytochrome pathway will always operate faster than the alternative pathway.

The observations in our expression system might be explained in terms of ‘tunnelling’.

In the situation where the two pathways are active simultaneously, the ‘slow’

dehydrogenase (SDH) has a preference for the ‘slow’ oxidising pathway (alternative

pathway), whereas the ‘fast’ dehydrogenase (external NADH dehydrogenase) has a

preference for the ‘fast’ oxidising pathway (cytochrome pathway). This would mean that

the Q-pool would be partitioned into functionally separate pools of ubiquinone. Perhaps in

the presence of two oxidases SDH in S. pombe mitochondria cannot keep the Q-pool

homogeneously reduced, a situation which would be exacerbated by deactivation of SDH

as occurs during deenergization of the IMM. It can however compete effectively with the

external NADH dehydrogenase for ubiquinone molecules, hence the strong control of SDH

on oxidising pathway kinetics as seen in figures 6.5 and 6.6.

Another explanation would be a non Q-dependent interaction between reducing and

oxidising pathways. It is known that AOX activity in Acanthamoeba castellanii

mitochondria is pH dependent [82, 258]. Importantly for this study, S. guttatum AOX also

shows such a dependency [83]. Possibly, in S. pombe mitochondria expressing AOX there

236

is a substrate dependent difference in matrix pH which could affect AOX activity. This

would only be apparent under the condition where both pathways are active at the same

time and this mechanism could be compatible with our results.

7.2.5 Future work suggestions pertaining to the substrate dependent oxidising pathway

kinetics in S. pombe mitochondria expressing AOX—In order to determine quantitatively

the distribution of reducing equivalents between the cytochrome pathway and the

alternative pathway with either succinate or NADH as a substrate there are at present two

approaches available. Under state 3 conditions, using the ADP/O method [267] one can

determine the distribution of reducing equivalents between the cytochrome and alternative

pathway. The method rests on the assumption that AOX activity does not contribute to ATP

formation and the ADP/O value in the presence of AOX is lowered as opposed to the

situation where it is inhibited (with OG for example) and can only be applied under state 3

conditions. This method, applied to S. pombe mitochondria expressing AOX could under

state 3 conditions quantitatively show the difference in engagement of the alternative and

the cytochrome pathway dependent on substrate.

Another method which has been used to quantify the distribution of reducing equivalents

between the alternative and the cytochrome pathway is the oxygen isotope discrimination

method [268]. The cytochrome and alternative pathway discriminate differently against the

isotope 18

O. Employing mass spectrometry it is feasible to asses the partition of reducing

equivalents between pathways in mitochondria. This technique however requires expensive

equipment and is not easy to use.

In our laboratory another alternative oxidase, from a non thermogenic species (Arabidopsis

thaliana) has been expressed successfully in S. pombe [70]. This system provides the

opportunity to answer the question whether or not substrate dependent differences are

specific to the S. pombe expression system in future work.

237

7.3 Conclusion

SDH regulation in plants appears to be more complicated than previously assumed. The

recent discovery of extra subunits make a direct comparison of results obtained in

mammalian and yeast systems with plant SDH difficult. Although SDH regulation has been

studied for over 50 years now it is clear that this subject is still as interesting as ever and

worthy of investigation.

Biphasic respiratory kinetics were an unexpected finding. Our results indicate that they may

be a general characteristic of yeast mitochondria, these results open new avenues of

research investigating the bc1 complex in mitochondria.

The expression system used in this laboratory has been and still is being used for the

investigation of structure-function relationships of the alternative oxidase. This study has

revealed some interesting findings which may shed more light on the process of AOX

regulation. The substrate dependent differences found could not be reproduced in a system

naturally expressing AOX, which may indicate that it is a characteristic of the system itself.

If that were the case, then our system is still very useful for the purpose of investigating

bioenergetic questions of a more fundamental nature, such as investigating the limitations

of the abilities of reducing pathways to keep the Q-pool homogenously reduced or

mechanisms via which reducing pathways can interact with oxidising pathways in a non Q-

dependent manner.

238

Appendix 1

Modelling of Q-pool data—The fits used to describe the Q-pool kinetics presented

throughout this thesis were modelled according to van den Bergen et al. [76]. In this model,

each of the Q(H2)-interacting pathways is assumed to exhibit reversible Michaelis-Menten

kinetics in accordance with the following scheme:

where the forward reaction represents the oxidation of quinol substrate (S) to give quinone

product (P) catalysed by enzyme (E). Described below is a derivation of a rate equation

which differs from the general reversible Michaelis-Menten equation in one aspect, namely

the sum of product and substrate is assumed to be constant (the ‘pool assumption’) as

opposed to the conventionally used initial conditions assumption.

The following definitions and assumptions apply:

[S] + [P] + [ES] = q

[E] + [ES] = e

[ES] e << [S], [P] so that with [S] = s and [P] = p

s + p = q

solution kinetics apply (rates are proportional to concentrations)

ES reaches a steady state

The steady state equation is:

[k1s + k4 p] [e – [ES]] = [k2 + k3] [ES] [1]

PE ES E S3

4

1

2

k

k

k

k

239

The rate (oxidation of ubiquinol) is defined as:

[2]

The net forward rate is:

v = k1se – [k1s + k2] [ES] = -k4pe + [k4p + k3] [ES] [3]

Eliminating ES yields:

[4]

This can be rearranged to:

[5]

Using common definitions for Ks, Kp, Vs and Vp:

Vs = k3e

4

32

k

kkKp

Vp = k2e

the rate equation becomes:

dt

Sdv

][

)()( 34

4

21

1

kpk

pekv

ksk

sekv

][][

][

4132

4231

pkskkk

epkkskkv

1

32

k

kkKs

240

[6]

Using the ‘pool assumption’ where s + p = q, p can be substituted for q – s which yields:

[7]

For fitting purposes it is convenient to use a simpler formula. By dividing both numerator

and denominator by (1 + q / Kp) the following equation is obtained:

[8]

With:

Kp

p

Ks

s

Kp

pVp

Ks

sVs

v

1

pps

p

p

p

p

s

s

K

qs

KK

K

qVs

K

V

K

V

v

111

v s

s 1

Vp

Kp 1

Kp

Ks

Vs Vp

Kp 1

Kp

Ks

1

Kp 1

241

Appendix 2A

Table

4.1A

including data on number of mitochondrial isolations and amount of experimental traces. Format: respiratory rate ( S.D.) [experimental traces, preps].

* SDH is activated stepwise by addition of ATP (0.2 mM) and glutamate (9 mM). vO2 with just succinate is

57 (7) [14,11] addition of ATP subsequently leads to a vO2 of 72 (12) [14,9]

A Respiratory Rate (nmol O2 min-1

mg-1

protein)


NADH 91 (13) [46,18] 281 (39) [20,8] 110 (22) [24,11] 537 (68) [23,14]

succinate *

106 (15) [19,11] 156 (18) [17,7] 97 (7) [16,6] 144 (25) [10,6]

glutamate 32 (4) [4,2] - - -

glutamate + ATP 43 (8) [4,2] - - -

242

Appendix 2B

sp.011 AOX Respiratory Rate (nmol O2 min-1

mg-1

protein)


NADH 199 (27) [37,14] 328 (34) [8,3] 224 (13) [3,1] 531 (82) [14,13]

NADH + OG 92 (10) [17,9] - - 396 (43) [19,9]

NADH + AA 81 (17) [16,13] - - -

succinate 142 (14) [17,4] 198 (13) [11,5] 136 (8) [11,4] 176 (15) [7,4]

succinate + OG 83 (12) [3,3] 123 (21) [3,3] 72 (6) [2,2] -

succinate + AA 84 (13) [10,4] - - -

sp.011 AOX + T

NADH 80 (13) [32,12] 169 (29) [7,4] 72 (9) [4,3] 398 (55) [23,11]

NADH + OG 77 (12) [15,8] - - 350 (48) [14,7]

succinate 83 (10) [16,5] 136 (16) [13,5] 87 (10) [11,5] 124 (20) [13,4]

sp.011 pREP

243

NADH 96 (13) [10,3] 245 (25) [8,3] - 543 (55) [10,3]

succinate 100 (13) [3,3] 196 (24) [5,3] 107 (23) [2,2] -

sp.011 wt

NADH 91 (13) [46,18] 281 (39) [20,8] 110 (22) [24,11] 537 (68) [23,14]

succinate 106 (15) [19,11] 156 (18) [17,7] 97 (7) [16,6] 144 (25) [10,6]

Table 5.2 including data on number of mitochondrial isolations and amount of experimental traces. Respiratory rates in sp.011 AOX, AOX + T, wt and pREP

mitochondria under various energetic conditions and various substrates. Format: substrate ( S.D.) [traces, preps]. Concentrations of chemicals added: NADH (1.8

mM), succinate (9 mM), octyl gallate (14 M), antimycin A (~40 nM). Uncoupled conditions are defined as respiratory activity in the presence of an uncoupler (2 µM

CCCP in all cases), which dissipates the protonmotive force leading to maximal activation of the cytochrome pathway. The concentration of added ADP to induce a

state 3 to state 4 transition was 0.2 mM in all cases. State 3 conditions were induced by pre-incubation of ADP (1 mM). All succinate rates are from mitochondria

incubated in the presence of ATP (0.2 mM) and glutamate (9 mM). The energy status had no effect on AOX activity, therefore the sp.011 AOX rates when incubated

in the presence of antimycin A are the same under all energetic conditions.

244

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