title: Nitroxides protect horseradish peroxidase from H2O2-induced inactivation and modulate its catalase-like activity.
abstract: Horseradish peroxidase (HRP) catalyzes H2O2dismutation while undergoing heme inactivation. The mechanism underlying this process has not been fully elucidated. The effects of nitroxides, which protect metmyoglobin and methemoglobin against H2O2-induced inactivation, have been investigated.
  
  
  
  HRP reaction with H2O2was studied by following H2O2depletion, O2evolution and heme spectral changes. Nitroxide concentration was followed by EPR spectroscopy, and its reactions with the oxidized heme species were studied using stopped-flow.
  
  
  
  Nitroxide protects HRP against H2O2-induced inactivation. The rate of H2O2dismutation in the presence of nitroxide obeys zero-order kinetics and increases as [nitroxide] increases. Nitroxide acts catalytically since its oxidized form is readily reduced to the nitroxide mainly by H2O2. The nitroxide efficacy follows the order 2,2,6,6-tetramethyl-piperidine-N-oxyl (TPO)>4-OH-TPO>3-carbamoyl proxyl>4-oxo-TPO, which correlates with the order of the rate constants of nitroxide reactions with compounds I, II, and III.
  
  
  
  Nitroxide catalytically protects HRP against inactivation induced by H2O2while modulating its catalase-like activity. The protective role of nitroxide at μM concentrations is attributed to its efficient oxidation by P940, which is the precursor of the inactivated form P670. Modeling the dismutation kinetics in the presence of nitroxide adequately fits the experimental data. In the absence of nitroxide the simulation fits the observed kinetics only if it does not include the formation of a Michaelis-Menten complex.
  
  
  
  Nitroxides catalytically protect heme proteins against inactivation induced by H2O2revealing an additional role played by nitroxide antioxidants in vivo.
authors.affiliation: Institute of Medical Research Israel-Canada, Medical School, The Hebrew University of Jerusalem, Jerusalem 91120, Israel.
authors.forename: Amram
authors.initials: A
authors.lastname: Samuni
authors.affiliation: Nuclear Research Centre Negev, Beer Sheva, Israel.
authors.forename: Eric
authors.initials: E
authors.lastname: Maimon
authors.affiliation: Institute of Chemistry, The Accelerator Laboratory, 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: D000975:Antioxidants / Q000494:pharmacology*; D002374:Catalase / Q000378:metabolism*; D002384:Catalysis; D006735:Horseradish Peroxidase / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D009589:Nitrogen Oxides / Q000494:pharmacology*; D020011:Protective Agents / Q000494:pharmacology
chemical_list: D000975:Antioxidants; D009589:Nitrogen Oxides; D020011:Protective Agents; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D002374:Catalase
keywords: Catalase-like activity; Compound II; Compound III; EPR; HRP; Heme inactivation; Kinetics; Suicide inactivation; TPO