Studies on Cytochrome c Peroxidase

Abstract

The stoichiometry of the reaction of cytochrome c peroxidase with hydrogen peroxide, and of the resulting Compound ES with both ferrocyanide and ferrocytochrome c as donor has been re-examined in detail over a wide pH range.It was confirmed that cytochrome c peroxidase and hydrogen peroxide react in a 1: 1 molar ratio, and that 2 molecules of either donor are required to reduce Compound ES completely.The re-examination of the change in absorbance on formation of Compound ES gave a more reliable value of the difference extinction coefficient than those previously reported.It was established that the reaction of Compound ES with donors occurs in at least two stages of comparable rates.Comparison of optical and electron paramagnetic resonance titration curves showed that the two oxidizing equivalents of Compound ES were associated with distinct and partially independent changes in the properties of the enzyme.The rate of reaction of Compound ES with ferrocyanide was measuredover the pH range 5 to 8, and the reaction was found to be biphasic.Several mechanisms were considered to account for these observations.All of the mechanisms involve an enzyme intermediate compound which is only one oxidizing equivalent above the native state.A theoretical analysis of the observed kinetics and titration curves showed that the data presently available do not unequivocally distinguish among all the proposed mechanisms.However, they strongly support a mechanism in which two one-oxidizingequivalent intermediates are at rapid equilibrium.One of the most interesting aspects of the chemistry of cytochrome c peroxidase (cytochrome c:HzOe oxidoreductase, EC

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Affiliated Institutions

• University of Pennsylvania US

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