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

Background Metmyoglobin (MbFe^III) reaction with H₂O₂ has been a subject of study over many years. H₂O₂ alone promotes heme destruction frequently denoted "suicide inactivation," yet the mechanism underlying H₂O₂ dismutation associated with MbFe^III inactivation remains obscure. Methods MbFe^III reaction with excess H₂O₂ 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 H₂O₂ depletion, O₂ evolution, spectral changes of the heme protein, and the fate of the nitroxide by EPR spectroscopy. Results The rates of both H₂O₂ dismutation and heme inactivation processes depend on [MbFe^III], [H₂O₂] and pH. Yet the inactivation stoichiometry is independent of these variables and each MbFe^III molecule catalyzes the dismutation of 50 ± 10 H₂O₂ 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 H₂O₂ and by nitroxide, respectively. Conclusions The nitroxide effects on H₂O₂ dismutation catalyzed by MbFe^III demonstrate that MbFe^IV=O reduction by H₂O₂ 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-H₂O₂ 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.