Cytochrome c peroxidase (CcP) is a detoxification enzyme, designed to maintain low levels of intracellular H2O2 levels by reducing the H2O2 to water. The catalytic mechanism couples the two- electron reduction of H2O2 to two single-electron-transfer steps involving ferrocytochrome c. CcP initially reduces H2O2 to water generating an enzyme intermediate called CcP compound I (CcP-I). CcP-I is oxidized two equivalents above the native FeIII state of the CcP with the two oxidized sites identified as an FeIV heme and an oxidized tryptophan radical. CcP-I is reduced back to the native FeIII state of CcP by two single-electron-transfer reactions with ferrocytochrome c. A second enzyme intermediate called CcP compound II (CcP-II) is produced during the reduction of CcP-I back to CcP. There are two forms of CcP-II, one that retains the FeIV heme group called CcP-IIF and one that retains the tryptophan radical called CcP-IIR. CcP-IIF and CcP-IIR appear to equilibrate via intra-molecular electron transfer but the rate of this step has been difficult to measure with certainty. One of the specific aims of this research proposal is to elucidate the involvement of the CcP-IIF/CcP-IIR equilibrium in the catalytic mechanism of CcP. Cytochrome c and CcP must form a complex in order to transfer electrons and the cytochrome c/CcP system provides an exceptional opportunity to explore details of protein-protein recognition as well as long-range electron transfer within dynamic, electrostatically stabilized protein-protein complexes. Structural aspects of the system are very well characterized with crystal structures available for 1:1 complexes of yeast iso-1 cytochrome c/CcP and horse cytochrome c/CcP, as well as for the individual proteins. A major goal of this research proposal is to determine the factors involved in binding of the two proteins and how these factors govern the specificity of the interaction as well as rapid turnover of the complex. Specificity and rapid turnover have opposite requirements with respect to binding affinity and the enzyme must evolve to optimize both properties simultaneously. The interaction between cytochrome c and CcP is mainly electrostatic in nature. Forty-six charge-reversal mutations on the surface of CcP have been constructed and characterized. The effect of the charge-reversal mutations on cytochrome c binding will be investigated using isothermal titration calorimetry and this data will be used to locate the primary and secondary binding domains. The formation and dissociation rates of the cytochrome c/CcP complex will be determined during the project.
One of the fundamental organizing principles of life is the requirement for the interaction of proteins with other biological molecules. The research described in this proposal is designed to explore details of protein-protein recognition in the cytochrome c/CcP system where both specificity and rapid turnover are required for function. The relevance of this research project to public health is to further our basic understanding of specific protein-protein interactions that could lead to the development of therapeutics to modulate aberrant protein-protein interactions involved in disease.
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