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Structure-Function Relationship of Mitochondrial Cytochrome C Oxidase: Redox Centres, Proton Pathways and Isozymes

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Structure-Function Relationship of Mitochondrial Cytochrome C Oxidase: Redox Centres, Proton Pathways and Isozymes Structure-function relationship of mitochondrial cytochrome c oxidase: redox centres, proton pathways and isozymes Raksha Jagdish Dodia Structural Molecular Biology University College London Submitted for the Degree of Doctor of Philosophy 2014 Supervisor: Prof. Peter R. Rich Internal examiner: Dr. Joanne Santini External examiner: Prof. Peter J. Nixon 1 Signed declaration I Raksha J. Dodia confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract Cytochrome c oxidase (CcO) reduces O2 to water with four electrons from cytochromes c2+ and four matrix protons. The energy released is conserved in the protonmotive force by translocation of four additional protons into the intermembrane space. Electrons are transferred via CuA, haem a, to the binuclear centre; haem a3 and CuB, where O2 is reduced. Four major aspects of its structure/function have been investigated in this study. Mid-infrared (IR) spectroscopy has been used to probe redox-induced structural changes. By using electrochemically-poised samples of cyanide- and carbon monoxide-ligated bovine CcO these redox-linked IR changes were shown to be linked primarily with CuA and haem a metal centre transitions with fewer changes associated with transitions in haem a3 and CuB. CcO contains a cross-linked Tyr-His which is believed to form a Tyr radical in the PM intermediate. In this work, electrochemical conditions to induce Tyr-His model compound radicals have been combined with IR spectroscopy to record IR reference spectra. This has aided tentative assignment of IR bands at 1572 cm-1 or 1555 cm-1 to . v8a(C-C), 1519 cm-1 to v7a(C-O ) to the phenoxyl radical and at 1336 cm-1 to a His ring stretch of the cross-linked structure in the PM state of bovine CcO. There is strong evidence from mutagenesis studies in bacterial CcOs that the well- conserved D channel is the proton translocation pathway. However, mutagenesis studies in a human/bovine hybrid CcO of an extensive hydrophilic H channel suggest that it fulfils this function, at least in mammalian CcOs. A structural model of Saccharomyces cerevisiae CcO produced by homology modelling indicates that it also contains an H channel (Maréchal, A., Meunier, B., Lee, D., Orengo, C. and Rich, P. R. (2012) Biochim. Biophys. Acta. 1817, 620-628). However, measurements of the H+/e- stoichiometry of a yeast H channel mutant (Q411L/Q413L/S458A/S455A) suggest it is not critical for proton translocation in yeast CcO. The nuclear-encoded subunit 5 of yeast CcO has two isoforms, 5A and 5B. COX5A is expressed aerobically and COX5B below 1 µM O2. They are reported to alter core catalytic activity; however comparisons were not strictly controlled. Here mutants were constructed where COX5B expression was controlled by the COX5A promoter yielding wild type levels of aerobically expressed 5B isozyme. Interestingly, this 5B isozyme exhibits the same catalytic activity and oxygen affinity as the 5A isozyme and the previously observed elevated activity must arise from a secondary effect. 3 Contents 1 Introduction _________________________________________________ 19 1.1 Electron transport chain __________________________________________ 20 1.2 Protonmotive force _______________________________________________ 25 1.3 Cytochrome c oxidase function ____________________________________ 26 1.4 Cytochrome c oxidase structure ___________________________________ 27 1.5 Overview of the electron transfer route _____________________________ 28 1.6 Subunit I structure ________________________________________________ 29 1.7 Subunit II and III structure _________________________________________ 32 1.8 Reaction mechanism _____________________________________________ 33 1.9 P and F transient intermediates ____________________________________ 34 1.9.1 Experimentally induced P and F states ____________________________________ 35 1.9.1.1 PM ________________________________________________________________ 36 1.9.1.2 PR ________________________________________________________________ 37 1.9.1.3 F _________________________________________________________________ 37 . 1.9.1.4 F (F dot) _________________________________________________________ 37 1.9.2 Detection of a radical species in PM _______________________________________ 38 1.10 Route for substrate protons ______________________________________ 39 1.11 Route for translocated protons ___________________________________ 39 1.12 Model for proton translocation via the D pathway __________________ 40 1.13 Controversial points on the D pathway ____________________________ 43 1.14 Model for proton translocation via the H pathway __________________ 44 1.15 Controversial points on the H pathway ____________________________ 47 1.16 Infrared spectroscopy on functional carboxyl groups ______________ 48 1.17 Yeast CcO as a model system ____________________________________ 49 1.18 Supernumerary subunits _________________________________________ 50 1.18.1 Bovine supernumerary subunit IV and yeast subunit 5 _____________________ 52 1.19 Aims ___________________________________________________________ 54 2 Materials and methods _______________________________________ 56 2.1 Sources of (bio) chemicals ________________________________________ 57 2.2 Yeast mutant generation __________________________________________ 57 2.3 Construction of 5A or 5B isozymes of yeast CcO ____________________ 58 2.4 Yeast cell growth and mitochondrial preparation ____________________ 59 2.5 Determination of protein content in yeast mitos _____________________ 60 2.6 Purification of yeast CcO __________________________________________ 61 2.6.1 Purification method 1 ___________________________________________________ 61 2.6.2 Purification method 2 ___________________________________________________ 62 2.6.3 Purification of yeast CcO in the presence of digitonin _______________________ 66 2.7 Purification of bovine CcO ________________________________________ 67 2.8 ATR-FTIR spectroscopy overview __________________________________ 68 4 2.9 Bovine CcO layer preparation for ATR-IR spectroscopy _____________ 69 2.10 ATR-FTIR spectroscopy with controlled electrochemistry __________ 71 2.11 Redox mediators ________________________________________________ 73 2.12 IR difference spectroscopy _______________________________________ 74 2.12.1 Measurement of IR difference spectra ___________________________________ 75 2.12.2 Preparation CN and CO bound CcO for IR measurements _________________ 75 2.13 Electrochemically induced IR difference spectra of Tyr and Tyr-His model compounds ______________________________________________________ 76 2.14 Perfusion-induced IR difference spectroscopy _____________________ 76 2.15 IR data manipulation _____________________________________________ 78 2.15.1 Baseline corrections ___________________________________________________ 78 2.16 Normalisation of redox spectra ___________________________________ 79 2.17 Gaussian calculated IR absorbance spectra _______________________ 80 2.18 Determination of pKa values of Tyr-His(trimethyl) __________________ 82 2.19 Oxygen electrode assay to determine turnover number ____________ 82 2.20 Determination of oxygen affinity __________________________________ 84 2.21 Membrane reconstitution of bovine CcO __________________________ 86 2.21.1 Optimisation of membrane reconstitution of yeast CcO ____________________ 87 2.21.2 Respiratory control ratio ________________________________________________ 87 2.21.3 Ferrocytochrome c preparation _________________________________________ 88 2.21.4 Proton/electron stoichiometry ___________________________________________ 88 2.22 Cyclic voltammetry ______________________________________________ 90 2.22.1 Cyclic voltammetry measurements ______________________________________ 93 3 Separation and redox linkage of IR signatures of bovine cytochrome c oxidase _______________________________________________ 94 3.1 Introduction ______________________________________________________ 95 3.2 Aims _____________________________________________________________ 96 3.3 Results __________________________________________________________ 97 3.3.1 Unligated CcO: FR – O difference spectra ________________________________ 97 3.3.2 CN-ligated CcO: MV-CN – O-CN difference spectra________________________ 98 3.3.3 In situ visible spectra ___________________________________________________ 99 3.3.4 CN-ligated CcO: FR-CN – O-CN difference spectra _______________________ 100 3.3.5 In situ visible spectra of unligated and CN-ligated CcO _____________________ 102 3.3.6 CN-ligated CcO: FR-CN – MV-CN difference spectra _____________________ 103 3.3.7 CN ligation to CcO: O-CN minus O spectra _______________________________ 104 3.3.8 Visible spectrum of CN ligation __________________________________________ 105 3.3.9 CO-ligated CcO: FR-CO – MV-CO spectra_______________________________ 106 3.3.10 CN-ligated and CO-ligated CcO: calculated (MV-CN – O-CN) minus (FR-CO - MV-CO) double difference spectra _______________________________________________ 107 3.3.11 Comparison of all redox difference spectra ______________________________ 108 3.3.12 Assigning IR changes in the 1700-1000 cm-1 range with specific metal centre redox transitions _______________________________________________________________ 109 3.4 Discussion ______________________________________________________ 110 3.4.1 FR minus O IR difference spectrum of unligated bovine CcO _______________ 110 3.4.2 Monitoring
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