Dodia, RJ; (2014) Structure-function relationship of mitochondrial cytochrome c oxidase: redox centres, proton pathways and isozymes. Doctoral thesis , UCL (University College London).

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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.

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