Reactivity of peroxynitrite versus simultaneous generation of ·NO and O2·^- toward NADH

The oxidation of NADH by peroxynitrite takes place indirectly via the radical intermediates formed during the self-decomposition of peroxynitrite, i.e., ·OH and ·NO₂, and the oxidation yield exceeds 29% at relatively high NADH concentrations. The efficiency of oxidation of NADH by peroxynitrite is hardly affected by the presence of bicarbonate at physiological pH, and is remarkably increased when authentic peroxynitrite is replaced by low and equal fluxes of ·NO and O₂·^-. We determined the rate constants for the reactions of NADH with ·OH, CO₃·^-, and ·NO₂ to be (2.0 ±0.2) x 10¹⁰, (1.4 ± 0.3) x 10⁹, and (4.0 ±2.0) x 10₃ M-^-1 s-^-1, respectively. We show that the reaction of NADH with ·OH in aerated solution does not form O₂·^-, whereas the other one-electron oxidants oxidize NADH to NAD·, which in turn very efficiently reduces oxygen to O₂·^-. These results suggest that at physiological pH the oxidation of NADH by peroxynitrite in the absence or presence of bicarbonate occurs mainly through the reactions of NADH with ·OH or CO₃·^-, which are formed in about equal yields. The oxidation of NADH by continuous generation of ·NO and 0₂·^- proceeds via a chain mechanism, and therefore, the oxidation yield increases upon decreasing the flux of the radicals, and is higher than that obtained with authentic peroxynitrite.