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4.2 Cytochrome C Oxidase

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4.2 Cytochrome C Oxidase Structural and functional characterization of cytochrome c oxidase, cytochrome bc1 complex and heme A synthase from Aquifex aeolicus Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften vorgelegt beim Fachbereich 14 Biochemie, Chemie und Pharmazie der Johann Wolfgang Goethe Universität in Frankfurt am Main, Deutschland von Hui ZENG aus Hubei, China Frankfurt am Main (2019) (D30) vom Fachbereich Biochemie, Chemie und Pharmazie der Johann Wolfgang Goethe Universität als Dissertation angenommen. Dekan: Prof. Dr. Clemens Glaubitz 1. Gutachter: Prof. Dr. Klaas Martinus Pos 2. Gutachter: Prof. Dr. Hartmut Michel Datum der Disputation: Die Doktorarbeit wurde vom Dezember 2014 bis zum Dezember 2019 unter Leitung von Prof. Dr. Hartmut Michel in der Abteilung für Molekulare Membranbiologie am Max-Planck-Institut für Biophysik in Frankfurt am Main durchgeführt. Eidesstattliche Erklärung Hiermit versichere ich, dass ich die vorliegende Arbeit selbstständig angefertigt habe und keine weiteren Hilfsmittel und Quellen als die hier aufgeführten verwendet habe. Hui ZENG Frankfurt am Main, den TO MY BELOVED FAMILY Publications 1. Guoliang Zhu*, Hui Zeng*, Shuangbo Zhang*, Jana Juli, Xiaoyun Pang, Jan Hofmann,Yan Zhang, Nina Morgner, Yun Zhu, Guohong Peng, Hartmut Michel, Fei Sun. 3.3 Å structure of hyperthermophilic respiratory complex III from Aquifex aeolicus reveals the mechanism of its thermal stability. (*: contributed equally. Angew. Chem. Int. Ed.. doi:10.1002/anie.201911554) 2. Hui Zeng, Guoliang Zhu, Shuangbo Zhang, Janosch Martin, Nina Morgner, Fei Sun, Guohong Peng, Hao Xie and Hartmut Michel. The isolated intact heme A synthase from Aquifex aeolicus is a trimer. (mbio, in revision) Table of contents Table of contents List of figures ............................................................................................................... III List of tables .................................................................................................................. V Zusammenfassung...................................................................................................... VII Summary .................................................................................................................... XV Abbreviations and Symbols ...................................................................................... XXI 1 Introduction ................................................................................................................. 1 1.1 The electron transport chain ......................................................................... 1 1.2 Heme-copper terminal oxidases ................................................................... 4 1.2.1 Classification of heme-copper terminal oxidases .............................. 4 1.2.2 Structure of heme-copper terminal oxidases...................................... 6 1.2.3 Major function of cytochrome c oxidase ........................................... 9 1.3 Cytochrome bc1 complex ........................................................................... 13 1.3.1 Structure of cytochrome bc1 complex .............................................. 14 1.3.2 Functional mechanism of the cytochrome bc1 complex .................. 17 1.4 Heme A synthase ........................................................................................ 18 1.5 A. aeolicus (VF5) ....................................................................................... 23 1.5.1 A. aeolicus respiration ...................................................................... 24 1.5.2 Electron transport in A. aeolicus ...................................................... 24 1.6 Aim of this work ......................................................................................... 25 2 Materials and Methods .......................................................................................... 27 2.1 Material ...................................................................................................... 27 2.1.1 Chemicals ......................................................................................... 27 2.1.2 Bacterial strains ................................................................................ 27 2.1.3 Plasmids ........................................................................................... 28 2.1.4 Bacterial growth media and solutions .............................................. 30 2.1.5 Enzymes, proteins, markers and kits................................................ 31 2.1.6 Chromatographic columns and matrices .......................................... 32 2.1.7 Databases, servers and software ...................................................... 33 2.2 Methods ...................................................................................................... 34 2.2.1 Molecular Biology ........................................................................... 34 2.2.2 Protein expression and isolation ...................................................... 44 2.2.3 Protein characterization methods ..................................................... 53 2.2.4 Protein Crystallization ..................................................................... 59 2.2.5 Electron microscopy ........................................................................ 60 I Table of contents 2.2.6 Activity assay ................................................................................... 62 3 Results ................................................................................................................... 63 3.1 Characterization of AaCcO ........................................................................ 63 3.1.1 Heterologous expression and purification of AaCcO and cytochrome c555...................................................................................................... 63 3.1.2 Purification of AaCcO from native membranes ............................... 73 3.1.3 Functional analysis of AaCcO ......................................................... 74 3.1.4 Structure of AaCcO .......................................................................... 75 3.2 Characterization of Aabc1 ........................................................................... 85 3.2.1 Purification of Aabc1 from native membranes ................................. 85 3.2.2 Functional analysis of Aabc1 ............................................................ 86 3.2.3 Structure of Aabc1 ............................................................................ 87 3.3 Characterization of AaHAS ...................................................................... 102 3.3.1 Heterologous expression and purification of AaHAS .................... 102 3.3.2 Oligomerization of AaHAS............................................................ 106 3.3.3 Mutations of AaHAS...................................................................... 110 3.3.4 Structure of AaHAS ....................................................................... 112 4. Discussion ........................................................................................................... 127 4.1 Respiratory chain complexes of A. aeolicus ............................................ 127 4.2 Cytochrome c oxidase .............................................................................. 128 4.3 Cytochrome bc1 complex ......................................................................... 133 4.4 Heme A synthase ...................................................................................... 136 4.4.1 Oligomerization of AaHAS............................................................ 136 4.4.2 Structure of AaHAS ....................................................................... 139 5. References ........................................................................................................... 144 Appendix .................................................................................................................... 163 Acknowledgements .................................................................................................... 169 II List of figures List of figures Figure 1-1: Schematic diagram of the mitochondrial electron transport chain ...... 2 Figure 1-2: X-ray structures of HCOs .................................................................... 8 Figure 1-3: The structure of NORs ......................................................................... 9 Figure 1-4: Catalytic cycle of cytochrome c oxidase ............................................ 10 Figure 1-5: Scheme of electron transfer in ba3-CcO ............................................ 12 Figure 1-6: Structures of mitochondrial and bacterial cytochrome bc1 complex . 16 Figure 1-7: Sequence alignments of Qi pocket residues of cyt. b subunits from different species ............................................................................................ 17 Figure 1-8: Modified Q cycle mechanism ............................................................ 18 Figure 1-9: Heme A biosynthesis pathway ........................................................... 19 Figure 1-10: Membrane topology models of heme A synthases from different classes. .......................................................................................................... 20 Figure 1-11: Overall structure of heme A synthase from B. subtilis (PDB:6A2J) .... ......................................................................................................................
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