Abstract
BackgroundHorseradish peroxidase (HRP) catalyzes H2O2 dismutation 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. MethodsHRP reaction with H2O2 was studied by following H2O2 depletion, O2 evolution and heme spectral changes. Nitroxide concentration was followed by EPR spectroscopy, and its reactions with the oxidized heme species were studied using stopped-flow. ResultsNitroxide protects HRP against H2O2-induced inactivation. The rate of H2O2 dismutation 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. ConclusionsNitroxide catalytically protects HRP against inactivation induced by H2O2 while 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. General SignificanceNitroxides catalytically protect heme proteins against inactivation induced by H2O2 revealing an additional role played by nitroxide antioxidants in vivo.
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