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On the ultrafast kinetics of the energy and electron transfer reactions in photosystem I [Elektronische Ressource] / vorgelegt von Chavdar Lyubomirov Slavov

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153 pages
On the ultrafast kinetics of the energy and electron transfer reactions in Photosystem I Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf vorgelegt von Chavdar Lyubomirov Slavov aus Kyustendil, Bulgarien Düsseldorf/Mülheim an der Ruhr, August 2009 aus dem Max-Planck-Institut für Bioanorganische Chemie, Mülheim an der Ruhr Gedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf Referent: Prof. Dr. Alfred R. Holzwarth Koreferent: Prof. Dr. Peter Westhoff Tag der mündlichen Prüfung: 9 Juli 2009 If you have an apple and I have an apple and we exchange apples then each of us will still have one apple. But if you have an idea and I have an idea and we exchange ideas then each of us will have two ideas. George Bernard Shawiii CHAPTER 1 1 INTRODUCTION 1 1.1 LIGHT-DEPENDENT PHASE OF OXYGENIC PHOTOSYNTHESIS 3 1.1.1 The photosynthetic electron transfer chain 3 1.1.2 Electron transfer reactions in PS II 4 1.1.3 Electron transfer between PS II and PS I. 4 1.1.4 Electron transfer reactions in PS I 5 1.1.5 ATP synthesis 5 1.2 LIGHT-HARVESTING PIGMENTS OF PS I 5 1.2.1 Chlorophylls 5 1.2.2 Carotenoids 7 1.2.3 Influence of the environment 7 1.
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On the ultrafast kinetics of the energy and electron
transfer reactions in Photosystem I






Inaugural-Dissertation



zur Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultät
der Heinrich-Heine-Universität Düsseldorf


vorgelegt von

Chavdar Lyubomirov Slavov
aus Kyustendil, Bulgarien








Düsseldorf/Mülheim an der Ruhr, August 2009 aus dem Max-Planck-Institut für Bioanorganische Chemie, Mülheim an der Ruhr























Gedruckt mit der Genehmigung der
Mathematisch-Naturwissenschaftlichen Fakultät der
Heinrich-Heine-Universität Düsseldorf




Referent: Prof. Dr. Alfred R. Holzwarth
Koreferent: Prof. Dr. Peter Westhoff


Tag der mündlichen Prüfung: 9 Juli 2009

If you have an apple and I have an apple
and we exchange apples then each of us will still
have one apple. But if you have an idea and I
have an idea and we exchange ideas then each
of us will have two ideas.
George Bernard Shaw
iii CHAPTER 1 1
INTRODUCTION 1
1.1 LIGHT-DEPENDENT PHASE OF OXYGENIC PHOTOSYNTHESIS 3
1.1.1 The photosynthetic electron transfer chain 3
1.1.2 Electron transfer reactions in PS II 4
1.1.3 Electron transfer between PS II and PS I. 4
1.1.4 Electron transfer reactions in PS I 5
1.1.5 ATP synthesis 5
1.2 LIGHT-HARVESTING PIGMENTS OF PS I 5
1.2.1 Chlorophylls 5
1.2.2 Carotenoids 7
1.2.3 Influence of the environment 7
1.3 FATE OF THE PIGMENT EXCITED STATE 8
1.3.1 Excitation energy transfer 8
1.3.2 Electron transfer 10
1.4 STRUCTURE OF PHOTOSYSTEM I 10
1.4.1 Structure of cyanobacterial Photosystem I 11
1.4.2 Structure of higher plant Photosystem I 14
1.5 LIGHT HARVESTING IN PHOTOSYSTEM I 16
1.5.1 Trapping models of light harvesting kinetics – general concepts 16
1.5.2 Trapping kinetics in Photosystem I 17
1.5.3 The role of antenna size and ′red′ chlorophylls in the trapping kinetics of Photosystem I 18
1.5.4 Electron transfer in the reaction center of Photosystem I 18
1.6 GOALS AND STRUCTURE OF THIS WORK 20
CHAPTER 2 23
MATERIALS AND METHODS 23
2.1 SAMPLES AND THEIR PREPARATION FOR THE EXPERIMENTS 24
2.1.1 Higher plant Photosystem I – core and intact complexes 24
2.1.2 Cyanobacterial Photosystem I – monomers and trimers 24
2.1.3 Algae Photosystem I – core and intact complexes (wild type and mutants) 24
2.1.4 Redox state of the Photosystem I complexes during the experiments 25
2.1.5 Sample protection from reactive oxygen species (ROS) 25
2.2 CALCULATION OF THE OPTIMAL EXCITATION CONDITIONS FOR PS I COMPLEXES. 25
2.3 EXPERIMENTAL METHODS 27
2.3.1 Time-resolved fluorescence 27
2.3.2 Ultrafast transient absorption 30
2.4 DATA ANALYSIS 32
2.4.1 Global analysis 33
2.4.2 Lifetime density analysis 34
2.4.3 Target analysis 34
2.4.4 Quality of the fit 37
2.4.5 Average lifetime of the excited state and scaling analysis 38
2.4.6 Calculation of the standard free energy 39
v CHAPTER 3 41
INSTALLATION AND DEVELOPMENT OF A SYNCHROSCAN STREAK CAMERA SYSTEM FOR SUB-PS
TIME-RESOLVED FLUORESCENCE MEASUREMENTS 41
3.1 GENERAL PRINCIPLES OF SC OPERATION 42
3.2 EXPERIMENTAL SET-UP 43
3.2.1 Excitation source 43
3.2.2 Optical pathway, sample box, sample holder 44
3.2.3 Fluorescence detection 45
3.3 DATA ACQUISITION MODE 46
3.3.1 Analog integration vs. photon counting 46
3.3.2 Photon counting and the Moiré effect 47
3.4 TIME LINEARITY AND RESOLUTION OF THE SYNCHROSCAN STREAK CAMERA 48
3.5 SWEEP VOLTAGE STABILITY – ORIGIN OF THE SIGNAL JITTER AND DRIFT 50
3.5.1 Signal drift components 50
3.5.2 Correction procedures 52
3.6 LONG FLUORESCENCE DECAYS AND THE BACK SWEEP PROBLEM 52
3.7 SPECTRAL SENSITIVITY CORRECTIONS 53
3.8 FINAL REMARKS 55
CHAPTER 4 57
TRAP-LIMITED CHARGE SEPARATION KINETICS IN PHOTOSYSTEM I COMPLEXES FROM HIGHER
PLANT 57
4.1 INTRODUCTION 58
4.2 MATERIALS AND METHODS 61
4.3 RESULTS 61
4.3.1 Global analysis 61
4.3.2 Target analysis 63
4.4 DISCUSSION 64
4.4.1 Reaction center kinetics 64
4.4.2 Energy transfer dynamics 65
4.4.3 Fluorescence spectra of the model compartments 66
4.4.4 Trap-limited kinetics in higher plant PS I 68
4.4.5 Nature of the 'red' Chls energy transfer 70
4.4.6 Influence of the 'red' Chls on the energy trapping process 70
4.5 CONCLUSIONS 71
4.6 SUPPLEMENTARY MATERIALS 72




vi CHAPTER 5 75
TRAPPING KINETICS IN ISOLATED CYANOBACTERIAL PS I COMPLEXES 75
5.1 INTRODUCTION 76
5.2 MATERIALS AND METHODS 78
5.3 RESULTS 79
5.3.1 Global analysis 79
5.3.2 Target analysis 80
5.4 DISCUSSION 81
5.4.1 Energy transfer 81
5.4.2 RC kinetics 82
5.4.3 Fluorescence spectra of the model compartments 83
5.4.4 Nature of the trapping kinetics 85
5.4.5 Influence of the 'red' Chls on the trapping kinetics 86
5.5 CONCLUSIONS 87
CHAPTER 6 89
INTRA-ANTENNA ENERGY TRANSFER AND TRAPPING PROCESSES IN THE INTACT PHOTOSYSTEM I
COMPLEX OF CHLAMYDOMONAS REINHARDTII 89
6.1 INTRODUCTION 90
6.2 MATERIALS AND METHODS 91
6.2.1 Preparation of PS I samples 91
6.2.2 Sub-ps synchroscan streak camera fluorescence measurements 92
6.3 RESULTS 93
6.3.1 Kinetic modeling 93
6.4 DISCUSSION 95
6.4.1 Optimal number of antenna compartments and the excitation vector 95
6.4.2 Choice of excitation vector 96
6.4.3 The energy trapping kinetics 96
6.4.4 Spectral properties of the kinetic compartments 97
6.5 CONCLUSIONS 98
6.6 SUPPORTING MATERIALS 99
CHAPTER 7 103
PRIMARY ELECTRON TRANSFER IS INITIATED SEPARATELY IN TWO BRANCHES IN THE REACTION
CENTER OF PHOTOSYSTEM I 103
7.1 INTRODUCTION 104
7.2 RESULTS 107
7.2.1 Lifetime density analysis 107
7.2.2 Kinetic modeling 108
7.3 DISCUSSION 109
7.3.1 The initial CS event 109
vii 7.3.2 Uni- vs. bi-branched kinetic models 109
7.3.3 Assignment of the radical pairs and their spectra 111
7.3.4 Branching ratio 112
7.3.5 Free energy differences 112
7.3.6 The intrinsic CS rate as a universal property 113
7.3.7 Evolutionary conservation of bi-directionality 113
7.3.8 Implications for artificial photosynthetic systems 114
7.4 MATERIALS AND METHODS 114
7.5 SUPPORTING INFORMATION 115
7.5.1 Materials and Methods 115
7.6 SUPPORTING FIGURES 118
7.7 SUPPORTING TABLES 121
SUMMARY 123
ZUSAMMENFASSUNG 127
REFERENCES 131
LIST OF ACTIVITIES 141
ACKNOWLEDGMENTS 143

viii Abbreviations

A primary electron acceptor chlorophyll(s) in Photosystem I 0
ADP Adenosine diphosphate
A. thaliana Arabidopsis thaliana
ATP Adenostriphosphate
β(α)-DM n-dodecyl-β(α)-D-maltoside
C. reinhardtii Chlamydomonas reinhardtii
Car Carotenoid
CFD constant-fraction discriminator
Chl Chlorophyll
CS charge separation
Cyt cytochrome
DAS decay-associatedspectrum
DCM 4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran
Phe or F phenylalanine
ET energy transfer
EPR electronparamagnetic resonance
Fd ferredoxin
+ reductase FNR ferredoxin-NADPH
FWHM full-width at half-maximum
IRF instrument response function (=PR)
LHC I light-harvesting complex I of green plants
LHC II ex II of green plants
Lut lutein
MCP microchannel plate
MES 2-(N-morpholino)ethanesulfonic acid
Met or M methionine
MO molecular orbital
NADPH nicotinamide adenine dinucleotide phosphate
OD optical density
OEC oxygen-evolving complex
OPA optical parametric amplifier
OPO optical parametric oscillator
PC plastocyanin
PD photodiode
PMS phenazine methosulphate
Phe or F phenylalanine
PQ plastoquinone
PS I Photosystem I
PS II Photosystem II
PR prompt response (= IRF)
RC reaction center
RP radical pair
SAES species-associated emission spectrum
SADS species-associated absorption difference spectrum
SC streak camera
SPT single photon timing
TA transient absorption
TAC time-to-amplitude converter
TCSPC time-correlated single photon counting
SPT single photon timing
T. elongatus Thermosynechococcus elongatus
TMH transmembrane helix
Tyr or Y tyrosine
Vio Violaxanthin
WT wild type
ix