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 strenght. 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 μM) are nonhyperbolic. The rate expression requires squared terms in the 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 106 M−1 s−1 at 300 mM ionic strength to 108 M−1 s−1 at 20 mM ionic strength and extrapolates to 5 × 108 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. © 1990, American Chemical Society. All rights reserved.