Effect of single-site charge-reversal mutations on the catalytic properties of yeast cytochrome c peroxidase: mutations near the high-affinity cytochrome c binding site.
Fifteen single-site charge-reversal mutations of yeast cytochrome c peroxidase (CcP) have been constructed to determine the effect of localized charge on the catalytic properties of the enzyme. The mutations are located on the front face of CcP, near the cytochrome c binding site identified in the crystallographic structure of the yeast cytochrome c-CcP complex [Pelletier, H., and Kraut, J. (1992) Science 258, 1748-1755]. The mutants are characterized by absorption spectroscopy and hydrogen peroxide reactivity at both pH 6.0 and 7.5 and by steady-state kinetic studies using recombinant yeast iso-1-ferrocytochrome c(C102T) as a substrate at pH 7.5. Some of the charge-reversal mutations cause detectable changes in the absorption spectrum, especially at pH 7.5, reflecting changes in the equilibrium between penta- and hexacoordinate heme species in the enzyme. An increase in the amount of hexacoordinate heme in the mutant enzymes correlates with an increase in the fraction of enzyme that does not react with hydrogen peroxide. Steady-state velocity measurements indicate that five of the 15 mutations cause large increases in the Michaelis constant (R31E, D34K, D37K, E118K, and E290K). These data support the hypothesis that the cytochrome c-CcP complex observed in the crystal is the dominant catalytically active complex in solution.
Pearl
Naw May
NM
Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, Illinois 60115, USA.
Jacobson
Timothy
T
Arisa
Moraa
M
Vitello
Lidia B
LB
Erman
James E
JE
D001665:Binding Sites; D002384:Catalysis; D003578:Cytochrome-c Peroxidase / Q000737:chemistry* / Q000235:genetics; D045304:Cytochromes c / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D006861:Hydrogen Peroxide / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D013050:Spectrometry, Fluorescence
D005656:Fungal Proteins; D045304:Cytochromes c; D006861:Hydrogen Peroxide; D003578:Cytochrome-c Peroxidase