Erman, James E.
Ph.D. (Doctor of Philosophy)
Department of Chemistry and Biochemistry
The interaction between cytochrome c peroxidase (CcP) and cytochrome c is investigated through the characterization of charge-reversal m utations on CcP. Fortyfour point mutants involve converting an acidic residue to a lysine residue, while two mutants involve positive-to-negative charge reversal, R31E and K149D. The mutants are characterized by uv-visible spectroscopy. The ratios of the absorbance in the Soret band to that in the protein band near 280 nm, A[sub soret]/A₂₈₀, are comparable to those of wild-type enzyme, with few exceptions. Many of the chargereversal mutations induce changes in the heme coordination, from predominantly penta-coordinate heme as found in wild-type CcP to predominantly hexa-coordinate heme in D37K, K149D, D241K, and E267K. At pH 7.5, the fraction of hexacoordinated heme increases for all mutants, indicating a pH -dependent shift in the binding of the sixth ligand. Analyses of the circular dichroic spectra for representative samples indicate that neither single-site mutations nor changes in heme coordination produce significant changes in the secondary structure of the proteins. Determination of the rates of the hydrogen peroxide reactions from stopped-flow studies indicate that penta-coordinate mutants have bimolecular rate constants similar to the wild type enzyme, while increases in hexa-coordination produce species which are less reactive toward hydrogen peroxide. Steady-state kinetic studies of the H₂O₂ oxidation of yeast cytochrome c catalyzed by CcP and its mutants yield the maximum turnover number, V[sub max]/e₀, and the Michaelis constant, KM. Values of V[sub max]/e₀ show a general correlation with the fraction of active enzyme, with turnover numbers decreasing with decreases in the fraction of active enzyme. The Michaelis constant, Kₘ, correlates well with the equilibrium constant for protein-protein binding. Five mutants, R3 IE, D34K, D37K, E118K, and E290K, show at least a twenty-four-fold increase in Kₘ, indicating a decrease in affinity for cytochrome c in these mutants. These five mutants are clustered at or near the crystallographically determined binding site. These results indicate the presence of a single cytochrome c binding domain on the surface of CcP.
Pearl, Naw May, "Cytochrome c binding to cytochrome c peroxidase : mutagenesis and kinetic studies" (2007). Graduate Research Theses & Dissertations. 2036.
xiii, 240 pages (some color pages)
Northern Illinois University
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Includes bibliographical references.