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UBC Theses and Dissertations

Functional properties of active site variants of yeast cytochrome C Rafferty, Steven Patrick


Mitochondrial cytochromes c are small, soluble respiratory proteins which exhibit a high degree of amino acid sequence and tertiary structural homologies across the entire eukaryotic kingdom. Theinfluence of mutations within two conserved regions of yeast-iso-1-cytochrome c on the physicochemical properties of this protein were examined. i) Invariant residue Phe82, located at the exposed heme edge on the protein surface, was replaced by Tyr, Leu, lie, Ala, Ser and Gly. ii) Internal residues Asn52, Tyr67, Thr78 and 1le75 form the environment about WAT166, a buried water molecule near the heme. This region undergoes relatively large oxidation-state linked conformational changes and these residues are collectively referred to as ‘water switch’ residues. The mutants Asn52Ala, Tyr67Phe, Ile75Met and Thr78Gly were studied. The effects of variation at these conserved positions on i) the oxidation-reduction equilibrium, ii) the electron transfer kinetics with inorganic electron transfer partners, and iii) the equilibrium and kinetics of heme axial ligand Met8O displacement by added azide and alkaline pH of yeast iso-1-cytochrome c, were determined. The reduction potentials (pH 6, 25 °C, μ=0.1M) of position 82 mutants decreased by up to 45 mV with decreasing size of the side chain. Small differences in the ΔH° and ΔS° of reduction were responsible for the observed reduction potentials. Electron transfer rates (corrected for driving force and electrostatics) with the inorganic electron transfer partners Fe(edta)^2- (as reductant) and Co(phen)₃ ³⁺ (as oxidant) increased up to 17-fold as the size of the mutant side chain decreased (pH 6, 25 °C, μ= 0.1M). Electron transfer reactivity relative to wild type was dependent on the identity of the inorganic electron transfer partner, indicating that mutations at position 82 change that portion of the surface of the eytochrome that contacts electron transfer partners. Eyring plots of the rate data showed small differences in the activation parameters of the mutants. [more abstract]

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