Cytochrome c peroxidase catalyzed oxidation of ferrocytochrome c by hydrogen peroxide: ionic strength dependence of the steady-state rate parameters.
The steady-state kinetics of the cytochrome c peroxidase catalyzed oxidation of horse heart ferrocytochrome c by hydrogen peroxide have been studied at both pH 7.0 and pH 7.5 as a function of ionic strength. Plots of the initial velocity versus hydrogen peroxide concentration at fixed cytochrome c are hyperbolic. The limiting slope at low hydrogen peroxide give apparent bimolecular rate constants for the cytochrome c peroxidase-hydrogen peroxide reaction identical with those determined directly by stopped-flow techniques. Plots of the initial velocity versus cytochrome c concentration at saturating hydrogen peroxide (200 microM) are nonhyperbolic. The rate expression requires squared terms in cytochrome c concentration. The maximum turnover rate of the enzyme is independent of ionic strength, with values of 470 +/- 50 s-1 and 290 +/- 30 s-1 at pH 7.0 and 7.5, respectively. The limiting slope of velocity versus cytochrome c concentration plots provides a lower limit for the association rate constant between cytochrome c and the oxidized intermediates of cytochrome c peroxidase. The limiting slope varies from 10(6) M-1 s-1 at 300 mM ionic strength to 10(8) M-1 s-1 at 20 mM ionic strength and extrapolates to 5 x 10(8) M-1 s-1 at zero ionic strength. The data are discussed in terms of both a two-binding-site mechanism and a single-binding-site, multiple-pathway mechanism.
Kim
K L
KL
Department of Chemistry, Northern Illinois University, DeKalb 60115.
Kang
D S
DS
Vitello
L B
LB
Erman
J E
JE
D000818:Animals; D001665:Binding Sites; D003574:Cytochrome c Group / Q000378:metabolism*; D003578:Cytochrome-c Peroxidase / Q000378:metabolism*; D006736:Horses; D006861:Hydrogen Peroxide / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D009206:Myocardium / Q000201:enzymology
D003574:Cytochrome c Group; D006861:Hydrogen Peroxide; D003578:Cytochrome-c Peroxidase