Mechanisms of Respiration

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DESCRIPTION (provided by applicant): Cytochrome c oxidase is one of the principal enzymes that provides energy for the cell. It does so by coupling dioxygen reduction to proton pumping and ultimately oxidative phophorylation. Cytochrome ba3 from Thermus thermophilus is a homolog that contains a storehouse of natural mutations that also endow the protein with novel chemical behaviors. These can be used to identify general features of energy transduction mechanisms. During past funding periods, we have developed expression systems for Thermus cytochrome c552, the natural substrate of ba3, and for ba3 itself. We also have in-house X-ray structures of both these molecules. These tools have permitted us to begin studies of the enzyme's mechanism. Our experimental approach involves study of the detailed chemistry of the Fea3-CuB site, exploration of the enzyme's mechanism, probing the roles of non-metal liganding amino acids close to CuB, and exploring the roles of amino acid residues, distant from the enzyme's redox centers, in proton transfer. There are four Specific Aims: [1] To determine the chemical nature of the Fea3-O-CuB bridge. [2] To determine the mechanism whereby cytochrome ba3 reduces dioxygen to water, including detailed information about electron transfer events, the chemical nature of Fea3-oxygen intermediates, their characteristic times of appearance and disappearance, and the corresponding dynamics and stoichiometry of proton exchanges during this process. [3] We will determine the role of the novel tyrosine (I-Y237) that is covalently linked to a histidine (I-H233) ligand to CuB. [4] We will use site-directed mutagenesis to assess the role of each of three novel proton transfer pathways suggested by the structure of cytochrome ba3. Throughout the proposed work, we use a wide variety of biochemical, spectral, and X-ray structural work to mount an aggressive, multi-pronged study of this important enzyme. The significance of this work to human health lies in providing fundamental information about the conservation of energy by biological systems.