Peroxynitrite-Mediated Heme Oxidation and Protein Modification of Native and Chemically Modified Hemoglobins

Peroxynitrite (ONOO−) has been shown to play a critical role in tissue reperfusion injury. We have studied the reactions of ONOO−with native and two chemically modified hemoglobins that are being developed as oxygen-carrying reperfusion agents for use in a variety of clinical conditions. Reactions of native and chemically modified oxyhemoglobins (oxyHb) at 7.4 with ONOO−lead to a rapid oxidation of the heme iron to ferric (HbFe3+) form. Addition of excess molar ratios of ONOO−to the ferryl (HbFe4+) heme protein induced a spectral change indicative of the reduction of HbFe4+to the HbFe3+oxidation state. No major spectral changes were noted when ONOO−was added to methemoglobin (HbFe3+) or cyanomethemoglobin (Hb3+CN−), whereas the carbonmonoxy derivative of ferrous hemoglobin (HbCO) underwent an immediate spectral change suggesting the displacement of the CO ligand and oxidation of the heme iron. Rapid mixing of ONOO−with oxyHb in the stopped-flow spectrophotometer yielded biphasic kinetic plots for the oxidation of the ferrous iron (Fe2+). Replots of the apparent rate constants for native, cross-linked and polymerized, cross-linked hemoglobins as a function of ONOO−concentration were linear, yielding a single second-order rate for all hemoglobins of between 2 to 3 × 104M−1s−1, independent of the oxygen affinities and molecular sizes of the proteins. Oxidative modifications of the protein by ONOO−, occuring primarily at the β subunits, were observed in reaction mixtures of oxyHb and ONOO−using reverse-phase HPLC. The immunodetection method confirms that nitration of tyrosine residues by ONOO−occurs on the hemoglobin molecule and contributes to the modifications observed. We postulate that the presence of hemoglobin in close proximity to ONOO−production sites in the vasculature can contribute to possiblein vivotoxicity by a two-step mechanism involving (i) direct oxidation of the heme iron and (ii) nitration of the tyrosine residues on the molecule, leading to subsequent instability and heme loss from the protein.