Erman, James E.
Ph.D. (Doctor of Philosophy)
Department of Chemistry and Biochemistry
This project assessed the reactivity of six site-specific, covalent, 1:1 cytochrome c-cytochrome c peroxidase (CcP) complexes using steady-state and transient-state kinetics. Five out of six covalent complexes, namely, CcP(Glu290Cys)-cytochrome c(Lys73Cys), CcP(Val5Cys)-cytochrome c(Lys79Cys), CcP(Lys264Cys)-cytochrome c(Lys79Cys), CcP(Lys12Cys)-cytochrome c(Lys79Cys), and CcP(Asn78Cys)-cytochrome c(Lys79Cys) were studied in depth. The CcP(Trp223Cys)-cytochrome c(Lys79Cys) complex was unable to react with hydrogen peroxide to form the oxidized intermediate called Compound I. The cytochrome c-CcP complexes catalyzed the steady-state oxidation of exogenous ferrocytochrome c upon initiation of reaction with hydrogen peroxide. The experiments were performed in 10 and 100mM potassium phosphate buffer, pH 7.5, 25°C. The ionic strength dependence of the steady-state parameters for the CcP(Val5Cys)-cytochrome c(Lys79Cys) complex were determined between 10 and 100mM ionic strength. The CcP(Glu290Cys)-cytochrome c(Lys79Cys) complex in which the crystallographically determined cytochrome c binding site is blocked with cytochrome c was found to be inert. These results strongly support the Single Electron Transfer Active Site mechanism. The plots of the initial velocities versus ferrocytochrome c concentrations at saturating hydrogen peroxide concentrations have been found to be hyperbolic for the remaining four complexes. The kcat values for all CcP-cytochrome c complexes, except CcP(Va15Cys)-cytochrome c(Lys79Cys) in 10mM potassium phosphate buffer, were either comparable to wild type CcP or smaller. The binding of exogenous cytochrome c occurs in the presence of a covalently bound cytochrome c if the covalently bound cytochrome c does not block the crystallographically defined binding site. The data show that the sites on CcP are interacting and the bound cytochromes c experience a strong electrostatic repulsion, especially at low ionic strength. This electrostatic interaction decreases with increasing ionic strength. The transient-state studies also point toward the sites being interacting since no detectable tryptophan radical species was observed in these experiments. Charge-charge repulsion between the positively charged covalently bound cytochrome c and the cationic radical at position 191 likely destabilizes the radical, causing the oxidized site to transfer to the heme iron.
Nakani, Siddhartha, "Synthesis and characterization of 1:1 covalent, site-specific cytochrome c peroxidase-cytochrome c complexes" (2005). Graduate Research Theses & Dissertations. 5173.
xii, 190 pages (some color pages)
Northern Illinois University
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