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Investigation of the light induced intermediate states in type I photosynthetic reaction center [Elektronische Ressource] / vorgelegt von Leysan Khuzeeva

202 pages
Investigation of the Light-Induced Intermediate States in Type I Photosynthetic Reaction CentersInaugural-DissertationzurErlangung des Doktorgrades derMathematisch-Naturwissenschaftlichen Fakultätder Heirich-Heine-Universität Düsseldorfvorgelegt vonLeysan Khuzeevaaus Kazan, Ru βlandDüsseldorf, April, 2009Aus dem Max-Planck-Institut für Bioanorganische Chemie, Mülheim an der Ruhr , GermanyGedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Heirich-Heine-Universität DüsseldorfReferent: Prof. Dr. Wolfgang LubitzKorreferent: Prof. Dr. Lutz SchmittTag der mündlichen Prüfung: 19 April, 2009For my familyAll truths are easy to understand once they are discovered; thepoint is to discover them. (Galileo Galilei)AcknowledgementsFirst of all I would like to thank my supervisor Professor Dr. Wolfgang Lubitz for providing me the possibility to work on an interesting PhD thesis. Vielen Dank!Prof. Dr. Lutz Schmitt is acknowledged for being the second reviewer of my thesis.I would like to thank my co-supervisor Dr. Mikhail L. Antonkine for his advices and help during my thesis work. I also knowledge Dr. Jens Niklas, Dr. Anton Savitsky, Dr. Alexey Silakov, Dr. Shipra Prakash, Dr. Özlen Erdem, Dr. Maria-Eirini Pandelia,Dr. Nicholas Cox and Prasanna Rangadurai for good discussions and valuable comments and remarks on my thesis work. I sincerely thank to Dr. Mikhail L. Antonkine and Dr.
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Investigation of the Light-Induced
Intermediate States in Type I
Photosynthetic Reaction Centers
Inaugural-Dissertation
zur
Erlangung des Doktorgrades der
Mathematisch-Naturwissenschaftlichen Fakultät
der Heirich-Heine-Universität Düsseldorf
vorgelegt von
Leysan Khuzeeva
aus Kazan, Ru βland
Düsseldorf, April, 2009Aus dem Max-Planck-Institut für Bioanorganische Chemie,
Mülheim an der Ruhr , Germany
Gedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen
Fakultät der Heirich-Heine-Universität Düsseldorf
Referent: Prof. Dr. Wolfgang Lubitz
Korreferent: Prof. Dr. Lutz Schmitt
Tag der mündlichen Prüfung: 19 April, 2009For my familyAll truths are easy to understand once they are discovered; the
point is to discover them. (Galileo Galilei)Acknowledgements
First of all I would like to thank my supervisor Professor Dr. Wolfgang Lubitz for
providing me the possibility to work on an interesting PhD thesis. Vielen Dank!
Prof. Dr. Lutz Schmitt is acknowledged for being the second reviewer of my thesis.
I would like to thank my co-supervisor Dr. Mikhail L. Antonkine for his advices and
help during my thesis work. I also knowledge Dr. Jens Niklas, Dr. Anton Savitsky,
Dr. Alexey Silakov, Dr. Shipra Prakash, Dr. Özlen Erdem, Dr. Maria-Eirini Pandelia,
Dr. Nicholas Cox and Prasanna Rangadurai for good discussions and valuable comments
and remarks on my thesis work. I sincerely thank to Dr. Mikhail L. Antonkine and Dr. Jens
Niklas for kindly providing the samples of PS I for the A project.0
I would like to thank all the Max Planck institute members who made the time spent for
me, especially Gudrun Klihm, Frank Reikowski, Christoph Laurich, Michael Reus, Horst
Selbach for their support with the experimental work.
I also would like Professor John H. Golbeck and their laboratory from the Department
of Biochemistry and Molecular Biology, The Pennsylvania State University, for providing
the samples of the heliobacterial reaction centers.
I would like to thank my friends and especially Dr. Ruslan Ovsyannikov for their he lp
and support. Thank You!
At last I would like to thank my parents and my husband Timur Khayrov for the ir
continuous help and giving motivation in difficult times of my thesis work and my life. Abstract
Photosynthesis is the natural process by which light energy is converted into
physiologically available chemical energy and is, thus, one of the most important processes in
nature. Light-induced charge separation between donor and acceptor molecules (cofactors)
initiates electron transfer in reaction center (RC). Subsequent electron transfer (ET) steps
proceed through a sequence of cofactors. Electron paramagnetic resonance (EPR)
spectroscopy allows the detection of intermediate states involved ine .g.E Tra (dicals, radical
pairs and triplet states). The focus of this work is EPR investigation of intermedia te electron
acceptors in Type I RC to obtain information about their structure and function.
Chapter 5 is devoted to the primary electron accept orin Athe Photosystem I (PS I). 0
The acceptor A is a chlorophyall (Chl a) molecule, as was shown by X-ray crystal structure 0
•+ •-
of PS I and optical data. The direct EPR investigation of trans iinent t heA P A radical 0 700 0
pair (RP) is difficult because of its short lifetime (~ 10 ns). Therefore, the intermediat e wAas 0
studied in the stationary photoaccumulated state. Additionally, the electrochemically
•-
generated Chla anion radical in liquid and frozen solution was used as model for the A0
system. Advanced EPR methods, such as ENDOR, TRIPLE, HYSCORE, were used to obtain
•-the hyperfine and quadrupole couplings of the photoaccumulated radica l aAnd the 0
electrochemically generated Chla anion radical. The comparison of the native cofactor and
the in vitro model system further clarified the current picture of the electronic structure of the
•-
primary electron acceptor A and the influence of the protein surrounding on the e lectron0
spin density distribution.
The Chapter 6 describes multifrequency EPR study of the primary photosynthetic
processes in the heliobacterial RC (HbRC). An investigation of HbRC is complicated due to
fast primary ET rates and the high sensitivity of the major pigment of HbRC, gBChl, to
oxidation. The main aim was to clarify the proposed role of a quinone as the secondary
electron acceptor A of HbRC, as is seen for PS I. Several biochemical treatments on HbRC 1
were performed, e.g. quinone replacement and photoaccumulation of ET cofactors. Transient
EPR (TREPR) spectroscopy was used to detect the short-lived paramagnetic states, i.e. RPs,
triplets. The influence of both oxidation and light excitation were investigated. The
explanation of the experimental results as well as parameters obtained from data analysis do
•+ •-not require the formation of the intermediate RP AP . However, the participation of the798 1
quinone in the ET of HbRC can not be explicitly excluded.Table of Contents
TABLE OF CONTENTS
ACKNOWLEDGEMENTS
ABSTRACT
TABLE OF CONTENTS
ABBREVIATIONS
CHAPTER 1 PHOTOSYNTHESIS
1.1 Importance of photosynthesis. . . . . . . . . . . . . .. 1. .
1.2 Brief history of discovery of photosynthesis . . . . . . . . . . . . 2
1.3 What happens during photosynthesis? . . . . . . . . . . . . . 4
1.4 Organization of photosynthetic organisms . . . . . . . . . . . 7
1.5 Type II reaction centers . . . . . . . . . . . . . .. 10. . .
1.6 Type I reaction centers: Photosystem I. . . . . . . . . . .. 12 . .
1.7 Antenna pigments in photosynthetic reaction centers . . . . . . . .. 16
Bibliography to Chapter 1 . . . . . . . . . . . . . . . 20. . .
CHAPTER 2 PRINCIPLES OF ELECTRON PARAMAGNETIC RESONANCE
2.1 Basic principles of electron paramagnetic resonance (EPR) . . . . . . . 31
2.2 Continuous wave (CW) and time-resolved (TR) EPR techniques . . . . . 34
2.3 Pulse EPR techniques . . . . . . . . . . . . . .. .35. . .
2.3.1 Electron Spin Echo Envelope Modulation (ESEEM) . . . . .. .36
2.3.2 HYperfine Sublevel CORrelation Spectroscopy (HYSCORE) . . . 39
2.3.3 Electron Nuclear Double Resonance (ENDOR) . . . . . . .. 40.
2.3.4 Electron Nuclear Nuclear Triple Resonance . . . . . . . .42 . .
Bibliography to Chapter 2 . . . . . . . . . . . . . . . 45. . .
CHAPTER 3 SPIN HAMILTONIAN OF THE TWO-SPIN SYSTEM
3.1 Spin Hamiltonian of the radical pair . . . . . . . . . . . .. 48.
3.2 Electron Zeeman Interaction . . . . . . . . . . . . . 50. . .
3.3 Zero-Field Splitting . . . . . . . . . . . . . . .. .52. . .
3.4 Nuclear Zeeman interaction . . . . . . . . . . . . .. 53 . . .
3.5 Hyperfine interaction . . . . . . . . . . . . . .. 53. . . .Table of Contents
3.6 Electron spin-spin interaction. . . . . . . . . . . . .. .55. .
3.7 Nuclear Quadrupole interaction . . . . . . . . . . . . 55. . .
Bibliography to Chapter 3 . . . . . . . . . . . . . . . 57. . .
CHAPTER 4 MATERIALS AND METHODS
4.1 Materials: Chlorophyll a anion radical in solution . . . . . . . . 58.
4.1.1 Chlorophyll a extraction and purification . . . . . . .. .58.
4.1.2 Electrochemical generation of Chl a anion radical . . . . .. .62
▪-
4.2 Materials: Primary electron acceptor A in Photosystem I . . . . .. .640
4.2.1 PS I samples . . . . . . . . . . . . . . .. 64. . . .
4.2.2Photoaccumulation experiments: preparation of stationary radicals in PS I . 64
4.3 Materials: Heliobacterial reaction centers fromHeliobac terium modesticaldum
(HbRC) . . . . . . . . . . . . . . . . . . . .65. . . .
4.3.1 Isolation and purification of HbRC . . . . . . . . . .. 65 . .
4.3.2 Oxygen sensitivity of HbRC . . . . . . . . . . . .66 . .
4.3.3 Quinone replacement . . . . . . . . . . . ...66. . .
4.3.4 Destruction of F . . . . . . . . . . . . . .. 67. . . .X
4.3.5 Photoaccumulation procedure . . . . . . . . . .. .67. .
4.4 Methods: Electron paramagnetic resonance techniques . . . . . . .. 68
4.4.1 CW and TREPR (X, Q, W-band setups) . . . . . . . . .68.
4.4.2 Pulsed EPR/ENDOR (X- and Q-bands setups) . . . . . . .. 70.
Bibliography to Chapter 4 . . . . . . . . . . . . . . .. 73. . .Table of Contents
CHAPTER 5 RESULTS AND DISCUSSION: PRIMARY ELECTRON
ACCEPTOR A IN PHOTOSYSTEM I: IN VITRO AND IN VIVO0
5.1 Introduction . . . . . . . . . . . . . . . .. .76. . . .
5.2 Results: Chlorophyll a anion radical in solution. . . . . . . . .. 80.
5.2.1 Electrochemistry of chlorophyll a . . . . . . . . . .. 80 . .
▪-5.2.2 CW EPR and ENDOR spectra of the chlorophyll a anion radical (Chl ) ina
liquid solution . . . . . . . . . . . . . . . . .82 . . . .
▪-
5.2.3Advanced EPR/ENDOR measurements on the Chl a in frozen solution . 85
•-
5.2.4 Nitrogen hyperfine and quadrupole couplings of the Chl a . . . .. 92
▪- 5.3 Results: Primary electron acceptor A in PS I . . . . . . . .. .95 .0
5.3.1 EPR measurements on photoaccumulated PS I samples . . . . .. 96
5.3.2 ENDOR measurements on photoaccumulated PS I samples . . . .. 101
▪- ▪-5.4 DFT calculations: electronic structure of Chl a and A radical . . . .. 1060
5.5 Summary and outlook . . . . . . . . . . . . . . .. 110. . .
Bibliography to Chapter 5 . . . . . . . . . . . . . . . 112. . .
CHAPTER 6 RESULTS AND DISCUSSION: CHARACTERIZATION OF HELIO-
BACTERIAL REACTION CENTERS: QUINONE ROLE IN ELECTR ON
TRANSFER?
6.1 Introduction . . . . . . . . . . . . . . . .. .116. . . .
6.2 Field dependence of spin polarized signals in HbRC . . . . . . . . . 123
6.3 Samples from different purification stages . . . . . . . . . . . 129
6.4 Proof of quinone participation in ET: chemical treatments . . . . . . . 132
6.4.1 Quinone replacement . . . . . . . . . . . ...132. . .
6.4.2 F destruction . . . . . . . . . . . . . . .. 137. . . .X
6.4.3 Investigation of photoaccumulated radicals in HbRC . . . . .. .138
6.5 Triplet states in HbRC . . . . . . . . . . . . . .. 141. . .
6.6 Oxygen sensitivity of HbRC . . . . . . . . . . . . .. .146. .Table of Contents
6.7 Action spectrum of HbRC. . . . . . . . . . . . . .. .149 . .
6.8 Charge recombination kinetics: temperature dependence. . . . . . . 151
6.9 Summary . . . . . . . . . . . . . . . ...158 . . . . .
Bibliography to Chapter 6 . . . . . . . . . . . . . . . 161. . . .
APPENDIX . . . . . . . . . . . . . . . . . . .172. . . . .
LIST OF FIGURES . . . . . . . . . . . . . . . . .177. . . .
LIST OF TABLES . . . . . . . . . . . . . . . . . 185. . . . .
CURRICULUM VITAE . . . . . . . . . . . . . . .. .187 . . . .