Reactivity of peroxynitrite versus simultaneous generation of (*)NO and O(2)(*)(-) 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 (*)NO(2), 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 O(2)(*)(-). We determined the rate constants for the reactions of NADH with (*)OH, CO(3)(*)(-), and (*)NO(2) to be (2. 0 +/- 0.2) x 10(10), (1.4 +/- 0.3) x 10(9), and (4.0 +/- 2.0) x 10(3) M(-)(1) s(-)(1), respectively. We show that the reaction of NADH with (*)OH in aerated solution does not form O(2)(*)(-), whereas the other one-electron oxidants oxidize NADH to NAD(*), which in turn very efficiently reduces oxygen to O(2)(*)(-). 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 CO(3)(*)(-), which are formed in about equal yields. The oxidation of NADH by continuous generation of (*)NO and O(2)(*)(-) 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.