Faculty of Sciences Institute of Plant Molecular Biology University of South Bohemia Czech Academy of Sciences PhD thesis Structure and function of pigment-protein complexes of photosynthetic microorganisms Miroslava Herbstová Supervisor: Doc. RNDr. František Vácha, PhD České Budějovice 2010 Herbstová, M., 2010: Structure and function of pigment-protein complexes of photosynthetic microorganisms. PhD thesis, in English – 113 p., Faculty of Science, University of South Bohemia, České Bud ějovice, Czech republic. Annotation Biochemical and biophysical methods in combination with electron microscopy were used to investigate structure and function of pigment-protein complexes in thylakoid membranes of various photosynthetic organisms. Prohlašuji, že jsem svoji diserta ční práci vypracovala samostatn ě pouze s použitím pramen ů a literatury uvedené v seznamu citované literatury. Prohlašuji, že v souladu s § 47b zákona č. 111/1998 Sb. v platném zn ění souhlasím se zve řejn ěním své diserta ční práce, a to v úprav ě vzniklé vypušt ěním vyzna čených částí archivovaných P řírodov ědeckou fakultou elektronickou cestou ve ve řejn ě p řístupné části databáze STAG provozované Jiho českou univerzitou v Českých Bud ějovicích na jejích internetových stránkách. V Českých Bud ějovicích, dne 31. kv ětna 2010 Miroslava Herbstová Acknowledgement First, I would like to express my gratitude to my supervisor František Vácha, for his encouragement and constant guidance during my PhD study. He was there to listen and give advice, review my work and proofread my papers. Certainly, he is responsible for helping me complete the writing of this dissertation. I also want to say a word of thanks to all members of Department of Photosynthesis of the Institute of Plant Molecular Biology, Biology Centre of the Academy of Sciences of the Czech Republic, particularly to Ivana Hunalová and František Matoušek for their technical support. I am grateful to my colleagues, Radek Litvín and David Bína for sharing their knowledge, experience and for interesting discussions. Last, but not least, I thank my parents for educating me and for unconditional support during the years. My special thanks belong to my mother for helping me at any time. Furthermore, I would also like to give thanks everyone who has not been mentioned and supported me anyway. Abbreviations A0 Primary electron acceptor in photosystem I, chlorophyll a molecule A1 Secondary electron acceptor in photosystem I, phylloquinone ADP Adenosine diphosphate APC Allophycocyanin ATP Adenosine triphosphate BChl Bacteriochlorophyll bH or bN High-potential heme of Cyt b6 close to negative side of membrane bL or bP Low-potential heme of Cyt b6 close to positive side of membrane Cab Chlorophyll a/b binding Car Carotenoid Car D1 , Car D2 β-carotene in D1 and D2 protein, respectively CF 0 Transmembrane component of chloroplast ATP synthase CF 0−CF 1 Chloroplast ATP synthase complex CF 1 Catalytic component of chloroplast ATP synthase Chl ZD1 , Chl ZD2 Peripheral chlorophyll of D1, D2 proteins, respectively Chl(s) Chlorophyll(s) cN Covalently bound heme of Cyt b6 close to a heme bN CP 43, CP 47 Core antenna proteins of photosystem II Cys Cysteine Cyt b6 Cytochrome b6 Cyt b6/f Cytochrome b6/f complex Cyt bc 1 Cytochrome bc 1 complex Cyt c6 Cytochrome c6 Cyt f Cytochrome f D1, D2 Photosystem II reaction center proteins of DGDG Digalactosyldiacylglycerol DNA Deoxyribonucleic acid FA, F B, F X Iron sulphur clusters of photosystem I FCP Fucoxanthin chlorophyll a/c protein Fd Ferredoxin FMO Fenna-Matthews-Olson BChl a protein of green sulphur bacteria FNR Ferredoxin:NADP + oxidoreductase GAP Glyceraldehyde-3-phosphate IsiA (CP 43') Iron Stress-Induced protein A; also called CP 43' LH1 Core light-harvesting complex of purple bacteria LH2 Accessory light-harvesting complex of purple bacteria LHC I Light-harvesting complex of PS I LHC II Light-harvesting complex of PS II LHC Light-harvesting complex Met Methionine MGDG Monogalactosyldiacylglycerol mRNA Messenger ribonucleic acid rRNA Ribosomal ribonucleic acid N Negative NADP + Nicotinamide adenine dinucleotide phosphate (oxidized) NADPH Nicotinamide adenine dinucleotide phosphate (reduced) OEC Oxygen-evolving complex P Positive P680 Primary electron donor in photosystem II absorbing light at 680 nm P700 Primary electron donor in photosystem I absorbing light at 700 nm PC Plastocyanin Pcb Prochlorophyte chlorophyll a/b binding protein PCH Phosphatidylcholine PCP Peridinin-chlorophyll a protein PCy Phycocyanin PE Phycoerythrin PEC Phycoerythrocyanin PG Phosphatidylglycerol PGA Phosphoglycerate Pheo Primary transient acceptor, pheophytin Pi Inorganic phosphate pmf Proton motive force PQ Plastoquinone PQ A Primary stable quinone electron acceptor of photosystem II PQ B Secondary stable quinone electron acceptor in photosystem II PQ BH2 Plastoquinol (fully reduced) PS I Photosystem I PS II core Core of the photosystem II PS II RC Photosystem II reaction centre (D 1/D 2/Cyt b559 /PsbI complex) PS II Photosystem II QN Plastoquinone-binding site in the cytochrome b6/f complex QP Plastoquinol-binding site in the cytochrome b6/f complex RC Reaction centre Rubisco Ribulose 1,5-bisphosphate carboxylase/oxagenase RuBP Ribulose 1,5-bisphosphate SDS−PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SQDG Sulphoquinovosyldiacylglycerol YD Tyrosine in position 161 in the D2 protein YZ Tyrosine in position 161 in the D1 protein β-Car β-carotene ∆ψ Transmembrane electric field Contents Chapter 1 ................................................................................................................................... 9 1 Introduction ..................................................................................................................... 10 1.1 Overview of photosynthesis ................................................................................... 10 1.2 Localization of photosynthesis .............................................................................. 11 1.2.1 Evolution of chloroplast ................................................................................. 11 1.2.2 Chloroplast – structure and organization ........................................................ 12 1.2.3 Lipid composition ........................................................................................... 14 1.3 Basic principles of photosynthesis ......................................................................... 15 1.3.1 Light absorption and energy delivery by antenna systems ............................. 16 1.3.2 Primary electron transfer in reaction centers .................................................. 16 1.3.3 Electron transport and generation of proton motive force .............................. 17 1.3.4 ATP synthesis ................................................................................................. 17 1.3.5 Carbon fixation and its incorporation into carbohydrates .............................. 18 1.4 Photosynthetic pigments ........................................................................................ 18 1.4.1 Chlorophylls ................................................................................................... 19 1.4.2 Carotenoids ..................................................................................................... 20 1.4.3 Phycobilins ..................................................................................................... 20 1.5 Photosynthetic apparatus ....................................................................................... 21 1.5.1 Photosystem II ................................................................................................ 22 1.5.2 Photosystem II function and electron transport pathway ............................... 27 1.5.3 Cytochrome b6/f complex ............................................................................... 29 1.5.4 Photosystem I ................................................................................................. 31 1.5.5 Photosystem I function and electron transport pathway ................................. 32 1.5.6 ATP synthase .................................................................................................. 33 1.5.7 Light-harvesting antenna complexes .............................................................. 34 1.6 Outline of the thesis ............................................................................................... 43 1.7 References .............................................................................................................. 44 Chapter 2 ................................................................................................................................. 59 2 Localization of Pcb antenna complexes in the photosynthetic prokaryote Prochlorothrix hollandica ............................................................................................. 60 2.1 Abstract .................................................................................................................. 60 Chapter 3 ................................................................................................................................. 62 3 Carotenoids in energy transfer and quenching processes in Pcb and Pcb–PS I complexes from Prochlorothrix hollandica ................................................................. 63 3.1 Abstract .................................................................................................................. 63 Chapter 4 ................................................................................................................................