The mechanism of oxidation of nitroalkanes by horseradish peroxidase.

Abstract

Horseradish peroxidase catalyzes the anaerobic oxidation of 2-nitropropane (probably as the nitronate R-) by H2O2 via Compounds I and II to form R-R. The oxidation rate is stimulated 10-fold by O2 and the products become acetone and NO-2. The aerobic oxidation of R- is a free radical chain reaction which is initiated by peroxidase and propagated by R because (a) R-R, reasonably, must arise as 2R leads to R-R, (b) over 90% of the enzyme-initiated reaction occurs free in solution, and (c) the Km value for R- is independent of the type of initiator. We present a scheme for initiation, propagation, and termination which explains product structure, the effects of resorcinol, CN-, ascorbate, superoxide dismutase, and catalase, as well as the ping-pong reaction kinetics. We used methanenitronate to investigate the locus of electron transfer from R- into the heme moiety of the enzyme because this donor reacts with Compound II to form an isoporphyrin, which then rearranges to a modified enzyme in which the ferriheme contains the nitromethyl group in covalent linkage at a methine carbon. The modified enzyme is 30-50% as active catalytically as the native enzyme. We argue that reduction of Compound II by R- occurs at the methine carbon by two competing pathways, namely, direct one electron transfer and, at about one-half the frequency, homolytic cleavage of a covalent heme-substrate adduct (isoporphyrin).