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 x 10(4) M-1 s-1, independent of the oxygen affinities and molecular sizes of the proteins. Oxidative modifications of the protein by ONOO-, occurring primarily at the beta subunits, were observed in reaction mixtures of oxyHb and ONOO- using reverse-phase HPLC. The immuno-detection 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 possible in vivo toxicity 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.