MECHANISM OF MANGANESE PEROXIDASE COMPOUND-II REDUCTION - EFFECT OF ORGANIC-ACID CHELATORS AND PH

The effect of oxalate, malonate, lactate, and succinate chelators on the reduction of Phanerochaete chrysosporium manganese peroxidase compound II by Mn-II was investigated using stopped-flow techniques. All rate data were collected from single-turnover experiments under pseudo-first-order conditions. With oxalate, the reduction of compound II by Mn-II exhibited saturation behavior when the observed pseudo-first-order rate constants were plotted against oxalate concentration. The plots passed through the origin, indicating that the reduction by Mn-II is irreversible at all concentrations of oxalate. Maximal stimulation of the rate of compound II reduction occurred at 2 mM oxalate, the concentration of oxalate found in the extracellular medium of agitated cultures of this fungus. In contrast, maximal stimulation of the reduction of compound II by Mn-II only was observed at high (>20 mM) nonphysiological concentrations of malonate and lactate. Furthermore, at low concentrations of malonate and lactate, the reduction of compound II appeared to be reversible. These results suggest that at physiological concentrations oxalate chelates and stabilizes Mn-III enhancing its efficient removal from the enzyme. The rate constants for compound II reduction exhibited bell-shaped curves as a function of pH and had optima at pHs 5.0-5.4. In the presence of succinate, triphasic kinetics were observed for compound II reduction by Mn-II. In contrast to the reduction of compound II by Mn-II, various chelators had no observable effect on the formation of compound I. However, they did affect the steady-state oxidation of 2,6-dimethoxyphenol.