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

• Published in: Biochimica et biophysica acta Vol. 1860; no. 7; pp. 1409 - 1416
• Main Authors: Samuni, Uri, Czapski, Gideon, Goldstein, Sara
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.07.2016
• Subjects: Catalase - chemistry; Catalase - metabolism; Catalysis; Compound II; Computer Simulation; Electron Spin Resonance Spectroscopy; Heme protein; Hydrogen Peroxide - chemistry; Hydrogen Peroxide - metabolism; Hydrogen-Ion Concentration; Kinetics; Mechanism; Metmyoglobin - chemistry; Metmyoglobin - metabolism; Models, Biological; Nitrogen Oxides - chemistry; Nitrogen Oxides - metabolism; Nitroxide; Oximetry; Suicide inactivation; Compound II; EPR; 4-OH-TPO; Heme protein; Compound III; Nitroxide; Mechanism; 3-CP; MbFeIII; NMR; PB; TPO; 4-oxo-TPO; Compound I; Suicide inactivation; Kinetics
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2016.04.002

Metmyoglobin (MbFeIII) 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 MbFeIII inactivation remains obscure. MbFeIII 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. The rates of both H2O2 dismutation and heme inactivation processes depend on [MbFeIII], [H2O2] and pH. Yet the inactivation stoichiometry is independent of these variables and each MbFeIII molecule catalyzes the dismutation of 50±10 H2O2 molecules until it is inactivated. The nitroxide catalytically enhances the catalase-like activity of MbFeIII while protecting the heme against inactivation. The rate-determining step in the absence and presence of the nitroxide is the reduction of MbFeIVO by H2O2 and by nitroxide, respectively. The nitroxide effects on H2O2 dismutation catalyzed by MbFeIII demonstrate that MbFeIVO 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 MbFeIVO and an inactivated heme at a ratio of 25:1. The effects of nitroxides are instrumental in elucidating the mechanism underlying the catalysis and inactivation routes of heme proteins. [Display omitted] •MbFeIII demonstrates low catalase-like activity while being inactivated.•The proposed mechanism involves the formation a compound I–H2O2 adduct.•Compound I–H2O2 decomposes to MbFeIVO and an inactivated form at a ratio of 25:1.•Nitroxides demonstrate that MbFeIVO reduction by H2O2 is the rate-determining step.•Nitroxides protects MbFeIII while enhancing its catalase-like activity.