Crystallographic and Mechanistic Studies of Cytochrome c Peroxidase

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The long term goal of this research is to understand the structure and catalytic mechanism of cytochrome c peroxidase (CCP). CCP is a 34,000 M.W. monomeric heme protein that catalyses the reductive cleavage of peroxide to water, using ferrous cytochrome c as the electron donor. The crystal structures of CCP, its two-electron oxidation product, Compound I (formed by the reaction of the enzyme with peroxide), enzyme:inhibitor complexes, and several mutant CCPs have been solved. The available structural information makes CCP an attractive system for the study of intermolecular electron transfer and O-O bond activation by heme proteins. The proposed investigation will employ site-directed mutagenesis to perturb the enzyme structure and X-ray crystallography to describe the resulting structural changes. Subsequent characterization of these mutants by spectroscopic and kinetic techniques will be used to relate the changes in structure to changes in the primary reactions of the enzyme. The results of the proposed research will provide insight into the structural features that are involved in electrostatic recognition between macromolecule and rapid transfer of electrons between heme proteins. The proposed work will also provide insight into the mechanisms that proteins use to modify the reactivity of the heme prosthetic group to accomplish a characteristic reaction.