Reaction of peroxynitrite with reduced nicotinamide nucleotides, the formation of hydrogen peroxide

NAD(P)H acts as a two-electron reductant in physiological, enzyme-controlle d processes. Under nonenzymatic conditions, a couple of one-electron oxidan ts easily oxidize NADH to the NAD(.) radical. This radical reduces molecula r oxygen to the superoxide radical (O-2(radical anion)) at a near to the di ffusion-controlled rate, thereby subsequently forming hydrogen peroxide (H2 O2), Because peroxynitrite can act as a one-electron oxidant, the reaction of NAD(P)H with both authentic peroxynitrite and the nitric oxide ((NO)-N-. ) and O-2(radical anion) releasing compound 3-morpholinosydnonimine N-ethyl carbamide (SIN-1) was studied. Authentic peroxynitrite oxidized NADH with a n efficiency of similar to 25 and 8% in the absence and presence of bicarbo nate/carbon dioxide (HCO3-/CO2), respectively. NADH reacted 5-100 times fas ter with peroxynitrite than do the known peroxynitrite scavengers glutathio ne, cysteine, and tryptophan, Furthermore, NADH was found to be highly effe ctive in suppressing peroxynitrite-mediated nitration reactions even in the presence of HCO3-/CO2. Reaction of NADH with authentic peroxynitrite resul ted in the formation of NAD(+) and O-2(radical anion) and, thus, of H2O2 wi th yields of about 3 and 10% relative to the added amounts of peroxynitrite and NADH, respectively. Peroxynitrite generated in situ from SIN-1 gave vi rtually the same results; however, two remarkable exceptions were recognize d. First, the efficiency of NADH oxidation increased to 60-90% regardless o f the presence of HCO3-/CO2, along with an increase of H2O2 formation to ab out 23 and 35%, relative to the amounts of added SIN-1 and NADH, Second, an d more interesting, the peroxynitrite scavenger glutathione (GSH) was neede d in a 75-fold surplus to inhibit the SIN-1-dependent oxidation of NADH hal f-maximal in the presence of HCO3-/CO2. Similar results were obtained with NADPH, Hence, peroxynitrite or radicals derived from it (such as, e.g. the bicarbonate radical or nitrogen dioxide) indeed oxidize NADH, leading to th e formation of NAD(+) and, via O-2(radical) (anion), of H2O2. When peroxyni trite is generated in situ in the presence of HCO3-/CO2, i.e. under conditi ons mimicking the in vivo situation, NAD(P)H effectively competes with othe r known scavengers of peroxynitrite.