Mechanistic studies of the oxidation of oxyhemoglobin by peroxynitrite

The strong oxidizing and nitrating agent peroxynitrite has been shown to diffuse into erythrocytes and oxidize oxyhemoglobin (oxyHb) to metHb. Because the value of the second-order rate constant for this reaction is on the order of 10(4) M(-)(1) s(-)(1) and the oxyHb concentration is about 20 mM (expressed per heme), this process is rather fast and oxyHb is considered a sink for peroxynitrite. In this work, we showed that the reaction of oxyHb with peroxynitrite, both in the presence and absence of CO(2), proceeds via the formation of oxoiron(iv)hemoglobin (ferrylHb), which in a second step is reduced to metHb and nitrate by its reaction with NO(2)(*). In the presence of physiological relevant amounts of CO(2), ferrylHb is generated by the reaction of NO(2)(*) with the coordinated superoxide of oxyHb (HbFe(III)O(2)(*)(-)). This reaction proceeds via formation of a peroxynitrato-metHb complex (HbFe(III)OONO(2)), which decomposes to generate the one-electron oxidized form of ferrylHb, the oxoiron(iv) form of hemoglobin with a radical localized on the globin. CO(3)(*)(-), the second radical formed from the reaction of peroxynitrite with CO(2), is also scavenged efficiently by oxyHb, in a reaction that finally leads to metHb production. Taken together, our results indicate that oxyHb not only scavenges peroxynitrite but also the radicals produced by its decomposition.