Pre-steady-state kinetics of intermediate formation in the deuteroferriheme-hydrogen peroxide system

Abstract

The pH dependence of formation of a peroxidatic intermediate from the reaction of deuteroferriheme with H2O2 was determined for the region pH 8.7-10.1 from stopped-flow kinetic studies in which absorbancy changes are observed at heme monomer-dimer isosbestic points. Results are interpreted primarily in terms of the attainment of double steady-state concentrations of Michaelis-Menton complex I and peroxidatic intermediate I''. A linear correlation of observed first-order rate constants with the fraction of deuteroheme existing as a monomer, the degree of dissociation of heme dimer, was demonstrated and nonzero intercepts were obtained. Slopes and intercepts show a linear logarithmic dependence on pH which is interpreted in terms of HO2- participation both in the formation and subsequent (catalatic) decomposition of a peroxidatically active intermediate. General acid catalysis of intermediate formation was indicated from studies in phosphate, arsenate and citrate buffer at pH 7.4-9.3. It was suggested that such catalysis may be responsible for anomalously high rates of H2O2 decomposition previously observed in phosphate buffer solution.