title: 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.
  
  
  CONCLUSIONS
  The effects of nitroxides are instrumental in elucidating the mechanism underlying the catalysis and inactivation routes of heme proteins.
authors.affiliation: Department of Chemistry and Biochemistry, Queens College, City University of New York, Flushing, NY 11367, USA.
authors.forename: Uri
authors.initials: U
authors.lastname: Samuni
authors.affiliation: The Accelerator Laboratory, Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
authors.forename: Gideon
authors.initials: G
authors.lastname: Czapski
authors.affiliation: The Accelerator Laboratory, Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel. Electronic address: sara.goldstein1@mail.huji.ac.il.
authors.forename: Sara
authors.initials: S
authors.lastname: Goldstein
mesh_terms: D002374:Catalase / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D003198:Computer Simulation; D004578:Electron Spin Resonance Spectroscopy; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008786:Metmyoglobin / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D009589:Nitrogen Oxides / Q000737:chemistry / Q000378:metabolism*; D010092:Oximetry
chemical_list: D009589:Nitrogen Oxides; D008786:Metmyoglobin; D006861:Hydrogen Peroxide; D002374:Catalase; C039900:nitroxyl
keywords: Compound II; Heme protein; Kinetics; Mechanism; Nitroxide; Suicide inactivation