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

作者：Samuni Uri; Czapski Gideon; Goldstein Sara^*

来源：Biochimica et Biophysica Acta-General Subjects, 2016, 1860(7): 1409-1416.

DOI：10.1016/j.bbagen.2016.04.002

• Heme protein
• Suicide inactivation
• Compound II
• Nitroxide
• Kinetics
• Mechanism

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 degrees C by direct determination of reaction rate constants using rapid-mixing stopped-flow technique, by following H2O2 depletion, O-2 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.

• 出版日期2016-7