Stopped-flow kinetic study of the H2O2 oxidation of substrates catalyzed by microperoxidase-8

We have studied the oxidation of microperoxidase-8 (MP-8) by H2O2 and the s ubsequent reaction of the intermediates with substrate by stopped-flow expe riments. Oxidation of MP-8 by H2O2 gives two intermediates, I and II. The o bserved rate constant for the formation of I is linearly dependent on [H2O2 ] and exhibits a bell-shaped dependence on pH with pK(a) values of 8.90 and 10.60, which are attributed to the deprotonation of MP-bound H2O2 and H2O, respectively. The observed rate constant for the conversion of I to II is independent of [H2O2], but increases sharply at pH > 9.0. The predominant f orms of the intermediate at pH 7.0 and 10.7 are I and II, respectively. Add ition of substrate to the intermediates at pH 9.0 gives rise to three disti nct stages, corresponding to the three steps (in decreasing order of rate): I --> II*, II --> MP, and II*--> MP. The rates of these steps are all line arly dependent on the substrate concentration and each individual rate cons tant has been determined. Substrate reactivity at pH 10.7 covers over two o rders of magnitude, ranging from 1.36 x 10(7) M-1 s(-1) for 1-naphthol to 4 .03 x 10(4) M-1 s(-1) for ferrocyanide. The substrate reactivity is linearl y correlated with its reduction potential, indicating that an electron tran sfer process is involved in the rate-limiting step.