Metmyoglobin and methemoglobin catalyze the isomerization of peroxynitrite to nitrate.

Hemoproteins, in particular, myoglobin and hemoglobin, are among the major targets of peroxynitrite in vivo. The oxygenated forms of these proteins are oxidized by peroxynitrite to their corresponding iron(iii) forms (metMb and metHb). This reaction has previously been shown to proceed via the corresponding oxoiron(iv) forms of the proteins. In this paper, we have conclusively shown that metMb and metHb catalyze the isomerization of peroxynitrite to nitrate. The catalytic rate constants were determined by stopped-flow spectroscopy in the presence and absence of 1.2 mM CO(2) at 20 and 37 degrees C. The values obtained for metMb and metHb, with no added CO(2) at pH 7.0 and 20 degrees C, are (7.7 +/- 0.1) x 10(4) and (3.9 +/- 0.2) x 10(4) M(-1) s(-1), respectively. The pH-dependence of the catalytic rate constants indicates that HOONO is the species that reacts with the iron(iii) center of the proteins. In the presence of 1.2 mM CO(2), metMb and metHb also accelerate the decay of peroxynitrite in a concentration-dependent way. However, experiments carried out at pH 8.3 in the presence of 10 mM CO(2) suggest that ONOOCO(2)(-), the species generated from the reaction of ONOO(-) with CO(2), does not react with the iron(iii) center of Mb and Hb. Finally, we showed that different forms of Mb and Hb protect free tyrosine from peroxynitrite-mediated nitration. The order of efficiency is metMbCN < apoMb < metHb < metMb < ferrylMb < oxyHb < deoxyHb < oxyMb. Taken together, our data show that myoglobin is always a better scavenger than hemoglobin. Moreover, the globin offers very little protection, as the heme-free (apoMb) and heme-blocked (metMbCN) forms only partly prevent nitration of free tyrosine.

Myoglobin scavenges peroxynitrite without being significantly nitrated.

We have analyzed in detail hemoglobin (Hb) and myoglobin (Mb) after treatment of different forms of these proteins with variable amounts of peroxynitrite. HPLC analyses of the peroxynitrite-treated proteins subjected either to acid hydrolysis or Pronase digestion showed that only very low quantities of 3-nitrotyrosine are formed when equivalent amounts of peroxynitrite are allowed to react with the oxy form of these proteins. Comparable amounts of nitrated amino acids are formed when metMb and metHb are treated with peroxynitrite under analogous conditions, but significantly larger yields are observed with apoMb and metMbCN. Interestingly, in addition we found that also the tryptophan residues of Mb and Hb are nitrated to a low but detectable extent. Taken together, our data suggest that the heme center of Mb may act as an efficient scavenger of peroxynitrite, protecting the globin from nitration. As peroxynitrite can irreversibly inhibit cytochrome c oxidase, oxyMb may utilize an additional important pathway to maintain mitochondrial respiration, that is, rapidly react with peroxynitrite and thus prevent nitration of other cellular components.