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

NAD(P)H acts as a two-electron reductant in physiological, enzyme-controlled processes. Under nonenzymatic conditions, a couple of one-electron oxidants easily oxidize NADH to the NAD(.) radical. This radical reduces molecular oxygen to the superoxide radical (O-2(radical anion)) at a near to the diffusion-controlled rate, thereby subsequently forming hydrogen peroxide (H2O2), 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-ethylcarbamide (SIN-1) was studied. Authentic peroxynitrite oxidized NADH with an efficiency of similar to 25 and 8% in the absence and presence of bicarbonate/carbon dioxide (HCO3-/CO2), respectively. NADH reacted 5-100 times faster with peroxynitrite than do the known peroxynitrite scavengers glutathione, cysteine, and tryptophan, Furthermore, NADH was found to be highly effective in suppressing peroxynitrite-mediated nitration reactions even in the presence of HCO3-/CO2. Reaction of NADH with authentic peroxynitrite resulted in the formation of NAD(+) and O-2(radical anion) and, thus, of H2O2 with yields of about 3 and 10% relative to the added amounts of peroxynitrite and NADH, respectively. Peroxynitrite generated in situ from SIN-1 gave virtually the same results; however, two remarkable exceptions were recognized. First, the efficiency of NADH oxidation increased to 60-90% regardless of the presence of HCO3-/CO2, along with an increase of H2O2 formation to about 23 and 35%, relative to the amounts of added SIN-1 and NADH, Second, and more interesting, the peroxynitrite scavenger glutathione (GSH) was needed in a 75-fold surplus to inhibit the SIN-1-dependent oxidation of NADH half-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 the formation of NAD(+) and, via O-2(radical) (anion), of H2O2. When peroxynitrite is generated in situ in the presence of HCO3-/CO2, i.e. under conditions mimicking the in vivo situation, NAD(P)H effectively competes with other known scavengers of peroxynitrite.