Reactivity of peroxynitrite versus simultaneous generation of (NO)-N-center dot and O-2(center dot-) toward NADH

The oxidation of NADH by peroxynitrite takes place indirectly via the radic al intermediates formed during the self-decomposition of peroxynitrite, i.e ., (OH)-O-. and (NO2)-N-., and the oxidation yield exceeds 29% at relativel y high NADH concentrations. The efficiency of oxidation of NADH by peroxyni trite is hardly affected by the presence of bicarbonate at physiological pH , and is remarkably increased when authentic peroxynitrite is replaced by l ow and equal fluxes of (NO)-N-. and O-2(.-). We determined the rate constan ts for the reactions of NADH with (OH)-O-., CO3.-, and (NO2)-N-. 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)-O-. in aerat ed 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 th rough the reactions of NADH with (OH)-O-. or CO3.-, which are formed in abo ut equal yields. The oxidation of NADH by continuous generation of (NO)-N-. and O-2(.-) proceeds via a chain mechanism, and therefore, the oxidation y ield increases upon decreasing the flux of the radicals, and is higher than that obtained with authentic peroxynitrite.