MUTAGENESIS ASSISTED FUNCTIONAL STUDIES OF CYTOCHROME C

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Three fundamental aspects of cytochrome c function that have general implications for many other proteins will be studied: (1) Regulation of cytochrome c conformational dynamics and midpoint reduction potential by pH. At alkaline pH, the Met80 ligand to the heme iron is replaced by either Lys79 or Lys73 to produce two alkaline conformational states. Electrochemical, stopped-flow, and NMR analysis of these two conformational isomers will be undertaken through analysis of the Lys79Ala and Lys73Ala variants to assess their structural and functional properties. Related studies will consider (a) the conformational dynamics of mutations at Phe82 that have previously been shown to lower the PKa for the conformational equilibrium, (b) the conformational and functional consequences of replacing residues 79 and 73 with alternative "alkaline ligands," and (c) the identity of the conformationally-linked titratable group that deprotonates prior to loss of the Met80 ligand. (2) Interaction and reaction of cytochrome c with other heme proteins. (a) The interaction of cytochrome c with cytochrome b5 will be studied through application of a novel NMR technique for identification of surface Lys residues involved in complex formation, through detailed electrostatics modelling of potentiometric titrations of the cytochrome c-cytochrome b5 complex and by rapid kinetics techniques. (b) The cytochrome c-cytochrome c peroxidase complex will be characterized by potentiometric titrations and rapid kinetics techniques that will include use of critically selected mutants of the peroxidase predicted to disrupt each of the two binding sites for the cytochrome. These results will also be simulated by electrostatics modelling calculations. (c) The complex formed by human erythrocyte cytochrome b5 and human hemoglobin and its subunits will be analyzed by potentiometric titrations. Parallel studies will assess the influence of complex formation on the spectroscopic and ligand binding characteristics of hemoglobin. (3) The role of electron donor structure on kinetics of intramolecular electron transfer kinetics. Synthetic flavocytochromes produced by a combination of site-directed mutagenesis and chemical modification will be used to study the effect of electron donor structure on intramolecular electron transfer kinetics by comparison with published results obtained with analogous cytochrome derivatives in which ruthenium complexes serve as the electron donor.