Kinetics of the reaction of compound II of horseradish peroxidase with hydrogen peroxide to form compound III

Abstract

The kinetics of the reaction of H2O2 with compound II of horseradish peroxidase were studied as a function of pH at 25 degrees C and constant ionic strength of 0.11 M. The reaction of H2O2 with compound II involves the transient formation of ferric peroxidase and superoxide anion as the first step followed by the reaction of the intermediate species with H2O2 to form compound III. Both reactions are also observed with peracetic acid as substrate, though the amplitude of the first step was too small for the rate to be measured. Observation of the first reaction was not possible below pH 8.5 under the conditions of this investigation. It tends to occur faster at lower pH so an increasing fraction is lost in the dead time of the stopped-flow apparatus. The rate constants for the second reaction, leading to compound III formation, are small at all pH values, with a maximum of 20 M-1 s-1 at pH 7.0. Groups on the enzyme intermediate species with pKa values of 4.2 and 9.1 appear to be involved in this reaction. Compound III formation is accompanied by oxidation of aromatic amino acid groups on the protein. The compound III formed from horseradish peroxidase compound II and hydrogen peroxide has bands with molar absorption coefficients in excellent agreement with those obtained by flash photolysis of aerated carbonmonoxyperoxidase [Wittenberg, J. B., Noble, R. W., Wittenberg, B. A., Antonini, E., Brunori, M. and Wyman, J. (1967) J. Biol. Chem. 242, 626-634]. Attempts to use m-chloroperbenzoic acid as oxidant resulted in the destruction of compound II.