Studies on Horseradish Peroxidase. XI. On the Nature of Compounds I and II as Determined from the Kinetics of the Oxidation of Ferrocyanide

Abstract

In order to investigate the nature of compounds I and II of horseradish peroxidase, the kinetics were studied of ferrocyanide oxidation catalyzed by these compounds which were prepared from three different oxidizing agents. The pH dependence of the apparent second-order rate constant for ferrocyanide oxidation by compound I, prepared from ethyl hydroperoxide and m-chloroperbenzoic acid, was interpreted in terms of an ionization on the enzyme with a pK_a = 5.3, identical to that reported previously for hydrogen peroxide. The second-order rate constant for the compound II-ferrocyanide reaction also showed the same pH dependence for the three oxidizing substrates. However, with more accurate results, the compound II-ferrocyanide reaction was reinterpreted in terms of a single ionization with pK_a = 8.5. The same dependence of ferrocyanide oxidation on pH suggests structurally identical active sites for compounds I and II prepared from the three different oxidizing substrates.