Samuni Uri, Czapski Gideon, Goldstein Sara
Department of Chemistry and Biochemistry, Queens College, City University of New York, Flushing, NY 11367, USA.
The Accelerator Laboratory, Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
Biochim Biophys Acta. 2016 Jul;1860(7):1409-16. doi: 10.1016/j.bbagen.2016.04.002. Epub 2016 Apr 6.
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.
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.
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.
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.
The effects of nitroxides are instrumental in elucidating the mechanism underlying the catalysis and inactivation routes of heme proteins.
高铁肌红蛋白（MbFe(III)）与H₂O₂的反应多年来一直是研究的课题。单独的H₂O₂经常促进血红素破坏，通常称为“自杀失活”，然而与MbFe(III)失活相关的H₂O₂歧化的潜在机制仍然不清楚。
在pH 5.3 - 8.1和25°C条件下，通过使用快速混合停流技术直接测定反应速率常数，通过跟踪H₂O₂消耗、O₂释放、血红素蛋白的光谱变化以及通过电子顺磁共振光谱法测定氮氧化物的命运，研究了在不存在和存在氮氧化物的情况下MbFe(III)与过量H₂O₂的反应。
H₂O₂歧化和血红素失活过程的速率均取决于[MbFe(III)]、[H₂O₂]和pH。然而失活化学计量与这些变量无关，并且每个MbFe(III)分子催化50±10个H₂O₂分子的歧化，直到其失活。氮氧化物催化增强MbFe(III)的过氧化氢酶样活性，同时保护血红素不被失活。在不存在和存在氮氧化物的情况下，速率决定步骤分别是H₂O₂和氮氧化物对MbFe(IV)O的还原。
氮氧化物对MbFe(III)催化的H₂O₂歧化的影响表明，H₂O₂对MbFe(IV)O的还原是该过程的速率决定步骤。所提出的机制充分符合氮氧化物的促催化和保护作用，意味着化合物I - H₂O₂加合物的中间体，其以25:1的比例分解为MbFe(IV)O和失活的血红素。
氮氧化物的作用有助于阐明血红素蛋白催化和失活途径的潜在机制。
