Reaction Mechanisms of Indole-3-acetate Degradation by Peroxidases. A Stopped-Flow and Low-Temperature Spectroscopic Study

Abstract

A study of reaction mechanisms of indole-3-acetate degradation by various peroxidases, in the absence of added hydrogen peroxide, has been performed employing stopped-flow and low-temperature spectroscopic techniques. At pH 6 and for high [substrate]/[enzyme] ratios, peroxidase compound II is formed and is stable for several minutes. However, this enzyme compound becomes visible only after the transient appearance of ferroperoxidase and of compound III. This reaction process is apparently linked with the formation of 3-methyleneoxindole as the major end-product of indole-3-acetate degradation. On the other hand, when indole-3-acetate and peroxidase in stoichiometric concentrations are mixed at pH 4, only ferroperoxidase and compound III are formed and the process appears to be linked with the production of indole-3-aldehyde. Stopped-flow experiments show that indole-3-acetate is rapidly bound on peroxidase, this rapid binding step being followed by a slow one-electron transfer between indole-3-acetate and peroxidase. This reduction of ferriperoxidase is blocked at low temperature (–20 °C), whereas in these experimental conditions, peroxidation of indole-3-acetate in the presence of added hydrogen peroxide still occurs. Moreover, when compound II reacts with indole-3-acetate at low temperature (–15 °C), this compound is regenerated as long as oxygen is present in the reaction medium. Neither ferroperoxidase nor compound I appear during this process. Most, if not all, of the compound III is formed by oxygenation of the ferrous peroxidase. This compound III can react with an equimolar amount of indole-3-acetate to give ferrous peroxidase and indole-3-aldehyde. Moreover, compound III can react with two indole-3-acetate molecules leading to compound II formation. Because only small amounts of the organic peroxide derived from indole-3-acetate are present in the reaction medium, most of the compound II is formed by a two-equivalent reduction of compound III, rather than through the direct interaction of the organic peroxide with peroxidase. Electronic mechanisms of reactions, consistent with the present experimental data, are proposed and discussed. They correlate the appearance and the disappearance of the various enzyme forms with known intermediates of indole-3-acetate degradation.