Characterization of the hydrogen peroxide-enzyme reaction for two cytochrome c peroxidase mutants.
The bimolecular reaction between Escherichia coli-produced cytochrome-c peroxidase (CcP(MI)) and hydrogen peroxide is identical to that of native yeast cytochrome-c peroxidase (CcP) and hydrogen peroxide in the neutral pH region. Both enzymes have pH-independent bimolecular rate constants of 46 microM-1.s-1 for the reaction with hydrogen peroxide. A second mutant enzyme, E. coli-produced cytochrome-c peroxidase mutant with phenylalanine at position 191 (CcP(MI, F191)), has a pH-independent bimolecular rate constant for the hydrogen peroxide reaction of 65 microM-1.s-1, 40% larger than for CcP or CcP(MI). The initial peroxide-oxidation product of CcP(MI, F191) is an oxyferryl porphyrin pi-cation radical intermediate in contrast to the oxyferryl amino-acid radical intermediate formed upon oxidation of CcP or CcP(MI) with hydrogen peroxide. The reactions of all three enzymes with hydrogen peroxide are pH-dependent in KNO3-containing buffers. The reactions are influenced by an ionizable group, which has an apparent pKa of 5.4 in all three enzymes. The enzymes react with hydrogen peroxide when the ionizable group is unprotonated. Both CcP(MI) and CcP(MI, F191) have slightly smaller pH stability regions compared to CcP as assessed by the hydrogen peroxide titer and spectral analysis. The alteration in structural stability must be attributed to differences in the primary sequence between CcP and CcP(MI) which occur at positions -2, -1, 53 and 152.
Vitello
L B
LB
Department of Chemistry, Northern Illinois University, DeKalb 60115.
Erman
J E
JE
Mauro
J M
JM
Kraut
J
J
D003578:Cytochrome-c Peroxidase / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005609:Free Radicals; D006861:Hydrogen Peroxide / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D010544:Peroxidases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship
D005609:Free Radicals; D006861:Hydrogen Peroxide; D010544:Peroxidases; D003578:Cytochrome-c Peroxidase