Nitroxide radicals as research tools: Elucidating the kinetics and mechanisms of catalase-like and "suicide inactivation" of metmyoglobin.

Abstract Background Metmyoglobin (MbFe III ) reaction with H 2 O 2 has been a subject of study over many years. H 2 O 2 alone promotes heme destruction frequently denoted “suicide inactivation,” yet the mechanism underlying H 2 O 2 dismutation associated with MbFe III inactivation remains obscure. Methods MbFe III reaction with excess H 2 O 2 in the absence and presence of the nitroxide was studied at pH 5.3–8.1 and 25 °C by direct determination of reaction rate constants using rapid-mixing stopped-flow technique, by following H 2 O 2 depletion, O 2 evolution, spectral changes of the heme protein, and the fate of the nitroxide by EPR spectroscopy. Results The rates of both H 2 O 2 dismutation and heme inactivation processes depend on [MbFe III ], [H 2 O 2 ] and pH. Yet the inactivation stoichiometry is independent of these variables and each MbFe III molecule catalyzes the dismutation of 50 ± 10 H 2 O 2 molecules until it is inactivated. The nitroxide catalytically enhances the catalase-like activity of MbFe III while protecting the heme against inactivation. The rate-determining step in the absence and presence of the nitroxide is the reduction of MbFe IV O by H 2 O 2 and by nitroxide, respectively. Conclusions The nitroxide effects on H 2 O 2 dismutation catalyzed by MbFe III demonstrate that MbFe IV O reduction by H 2 O 2 is the rate-determining step of this process. The proposed mechanism, which adequately fits the pro-catalytic and protective effects of the nitroxide, implies the intermediacy of a compound I–H 2 O 2 adduct, which decomposes to a MbFe IV O and an inactivated heme at a ratio of 25:1. General significance The effects of nitroxides are instrumental in elucidating the mechanism underlying the catalysis and inactivation routes of heme proteins.