Publication Date

2005

Document Type

Dissertation/Thesis

First Advisor

Erman, James E.

Degree Name

Ph.D. (Doctor of Philosophy)

Legacy Department

Department of Chemistry and Biochemistry

LCSH

Cytochrome c--Analysis

Abstract

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.

Comments

Includes bibliographical references (leaf 190).

Extent

xii, 190 pages (some color pages)

Language

eng

Publisher

Northern Illinois University

Rights Statement

In Copyright

Rights Statement 2

NIU theses are protected by copyright. They may be viewed from Huskie Commons for any purpose, but reproduction or distribution in any format is prohibited without the written permission of the authors.

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