Triplet states in bacterial reaction centers of Rhodobacter sphaeroides and related systems [Elektronische Ressource] / vorgelegt von Aliaksandr Marchanka

Marchanka

Sujets

• Biologie

Informations

• Publié par: heinrich-heine-universitat_dusseldorf
• Publié le: 01 janvier 2009
• Nombre de lectures: 21
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

Triplet states in bacterial reaction centers of
Rhodobacter sphaeroides and related systems
Inaugural-Dissertation
zur Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultät
der Heinrich-Heine-Universität Düsseldorf
vorgelegt von
Aliaksandr Marchanka
aus Orechowsk
Düsseldorf, Juni 2009Aus 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. W. Lubitz
Koreferent: Prof. Dr. K. Kleinermanns
Tag der mündlichen Prüfung: 26.06.2009 No llores porque ya se terminó… sonríe, porque sucedió.
To my mother and the memory of my father Aknowledgement
I would like to thank Dr. Maurice van Gastel, my supervisor, for his help, mentoring, support,
trust and patience in leading me through secrets of science. He is also thanked for carefully
reading and suggesting scientific and grammatical corrections of our publications and this
thesis.
Prof. Dr. Wolfgang Lubitz is gratefully acknowledged for giving me the opportunity to
perform my PhD project in his laboratory, for general supervising, and for being my first
reviewer. I would like to thank Prof. Dr. Karl Kleinermanns for being my second reviewer.
Dr. Jens Niklas, Dr. Shipra Prakash, Dr. Alexey Silakov and Maria Eirini-Pandelia are
gratefully acknowledged for the advices, fruitful discussions and help at various stages of this
project.
I would like to thank Gabriele Schmitz, Kathrin Schwarzbach, Michael Reus and Horst
Selbach for their help with preparations and purification of samples. Gudrun Klihm and Frank
Reikowski are gratefully acknowledged for the maintenance of EPR spectrometers and laser
systems.
Dr. M. Paddock and Prof. Dr. M. Y. Okamura are gratefully acknowledged for supplying Rb.
sphaeroides mutant samples and for discussions about our joint publication. I would like to
thank Dr. P. Gast for the Rb. sphaeroides quadruple mutant samples and Dr. A. Savitsky for
his help with the W-band measurements on this mutant.
Prof. Dr. E. J.J. Groenen, Prof. Dr. K. Möbius, Prof. H.-J. Steinhoff and Dr. M. Engelhard are
thanked for the collaboration within the DFG/NWO Teilprojekt GA1100/1-2. I would like to
thank the DFG, NWO, SFB 663, TP A7 and MPG for the financial support.
I would like to thank my friends Tatyana, Iryna, and Olga for their permanent support and
help. All my friends and colleagues are gratefully thanked for their help and advices.
At last but not least, I would like to express my sincerest gratitude to my dear mother for her
help, support, love and belief in me.
Table of contents
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii
Zusammenfassung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Abbreviations and symbols . . . . . . . . . . . . . . . . . . . . . . . . . vii
Chapter 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . 1
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Chapter 2. Bacterial reaction centers . . . . . . . . . . . . . 5
Structure and function of bacterial reaction centers . . . . . . . . . 5
The photosynthetic cofactors . . . . . . . . . . . . . . . . . . . . . 10
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Chapter 3. Theory and methodology . . . . . . . . . . . . . 17
Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
EPR spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Spin Hamiltonian . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Formation of the triplet state . . . . . . . . . . . . . . . . . . . . . 21
Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
cw EPR spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . 23
Pulsed EPR spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . 24
Quantum chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Chapter 4. Aims of the thesis . . . . . . . . . . . . . . . . . . . . . 33
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Chapter 5. Triplet state in bRC of Rhodobacter sphaeroides
and mutants studied by EPR . . . . . . . . . . . . . . . . . . . . . 37
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . 41
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
i Chapter 6. Spin density distribution of the triplet state of
bacteriochlorophylls . . . . . . . . . . . . . . . . . . . . . . . . . 69
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . 76
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Chapter 7. Spin density distribution of the triplet state of the
primary donor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . 105
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
Chapter 8. B-branch electron transfer in a quadruple mutant
of Rb. sphaeroides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . 131
Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . 132
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Chapter 9. Summary and outlook . . . . . . . . . . . . . . . . . 141
Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
ii Summary
In bacterial photosynthesis, the important process of light induced charge separation
occurs in photosynthetic reaction center proteins and proceeds from a primary donor (a dimer
of bacteriochlorophylls) predominantly along one of two nearly symmetric branches of
cofactors. The relative photosynthetic activity of the two branches can be advantageously
investigated by EPR and ENDOR spectroscopy of the triplet state of the primary donor.
Though the triplet state occurs with low yield in native systems, it can be induced to a yield of
unity by prereducing the final quinone electron acceptors. Since the paramagnetic triplet state
has the same electronic configuration as the diamagnetic photoexcited singlet state from
which charge separation initiates and since its polarization pattern depends on the presence or
absence of light-induced charge-separated intermediate states at either branch, the triplet EPR
spectrum of the primary donor contains essential information about the initial processes of
photosynthetic charge separation. In this thesis, the photoexcited triplet state of the primary
donor and the carotenoid in bacterial reaction centers of Rb. sphaeroides, in mutants, in
reaction centers of Bl. viridis and in model systems (bacteriochlorophylls in vitro), have been
investigated by EPR and ENDOR spectroscopy at cryogenic temperatures. The experiments
have been supplemented with DFT calculations.
The triplet state is found to derive from radical pair and intersystem crossing
mechanisms. The former mechanism is operative for Rb. sphaeroides wild type, R-26.1,
mutant GD(M203)/AW(M260) and Bl. viridis wild type in the measured temperature range 10 K –
100 K, indicating effective A-branch separation at these temperatures. The latter mechanism is
operative for bacteriochlorophylls in vitro and for Rb. sphaeroides mutants
LH(M214)/AW(M260) and HL(M182)/GD(M203)/LH(M214)/AW(M260). An intersystem
crossing triplet state indicates that no long-lived radical pairs are formed upon direct
excitation of the primary donor and that virtually no charge separation at the B-branch occurs
at low temperatu