The Mechanism of the Peroxynitrite-Mediated Oxidation of Myoglobin in the Absence and Presence of Carbon Dioxide

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Abstract
Among the cellular components that can react directly with peroxynitrite in the presence of physiological CO(2) concentrations are sulfur-, selenium-, and metal-containing proteins, in particular hemoproteins. We have previously shown that the reactions of peroxynitrite with oxymyoglobin (MbFeO(2)) and oxyhemoglobin proceed via the corresponding ferryl species, which, in a second step, are reduced to the iron(III) forms of the proteins (metMb and metHb). In this study, we have investigated the influence of the concentration of added CO(2) on the kinetics and the mechanism of the peroxynitrite-mediated oxidation of MbFeO(2). We found that this reaction proceeds in two steps via the formation of MbFe(IV)=O also in the presence of millimolar amounts of CO(2). As compared to the values measured in the absence of added CO(2), the second-order rate constant for the first reaction step in the presence of 1.2 mM CO(2) is significantly larger [(4.1 +/- 0.7) x 10(5) M(-1) s(-1), at pH 7.5 and 20 degrees C], whereas that for the peroxynitrite-mediated reduction of MbFe(IV)=O to metMb is almost unchanged [(3.2 +/- 0.2) x 10(4) M(-1) s(-1), at pH 7.5 and 20 degrees C]. Finally, we show that because of the parallel decay of peroxynitrite, 8-25 equiv are needed to completely oxidize MbFeO(2) to metMb, with larger amounts required to oxidize diluted MbFeO(2) solutions in the presence of CO(2). Simulation of the reaction in the absence and presence of CO(2) was carried out to get a better understanding of the mechanism. The results suggest that CO(3)*(-) and NO(2)* may be involved in the reaction and interact with MbFeO(2) and MbFe(IV)=O, respectively.