Peroxynitrite, the reaction product of nitric oxide (NO) and superoxide (O 2 . ) is assumed to decompose upon protonation in a first order process via intramolecular rearrangement to NO 3 ؊ . The present study was carried out to elucidate the origin of NO 2 ؊ found in decomposed peroxynitrite solutions. As revealed by stopped-flow spectroscopy, the decay of peroxynitrite followed firstorder kinetics and exhibited a pK a of 6.8 ؎ 0.1. The reaction of peroxynitrite with NO was considered as one possible source of NO 2 ؊ , but the calculated second order rate constant of 9.1 ؋ 10 4 M ؊1 s ؊1 is probably too small to explain NO 2 ؊ formation under physiological conditions. Moreover, pure peroxynitrite decomposed to NO 2 ؊ without apparent release of NO. Determination of NO 2 ؊ and NO 3 ؊ in solutions of decomposed peroxynitrite showed that the relative amount of NO 2 ؊ increased with increasing pH, with NO 2 ؊ accounting for about 30% of decomposition products at pH 7.5 and NO 3 ؊ being the sole metabolite at pH 3.0. Formation of NO 2 ؊ was accompanied by release of stoichiometric amounts of O 2 (0.495 mol/mol of NO 2 ؊ ). The two reactions yielding NO 2 ؊ and NO 3 ؊ showed distinct temperature dependences from which a difference in E act of 26.2 ؎ 0.9 kJ mol ؊1 was calculated. The present results demonstrate that peroxynitrite decomposes with significant rates to NO 2 ؊ plus O 2 at physiological pH. Through formation of biologically active intermediates, this novel pathway of peroxynitrite decomposition may contribute to the physiology and/or cytotoxicity of NO and superoxide.The reaction between nitric oxide (NO) and superoxide anion (O 2 . ) yields peroxynitrite with a second order rate constant near the diffusion-controlled limit (k ϭ 4.3-6.7 ϫ 10 9 M Ϫ1 s Ϫ1 ) (1, 2). The reaction constitutes an important sink for O 2 . because it is about twice as fast as the maximum velocity of SOD. 1 Consequently, peroxynitrite has been implicated in many pathological conditions including stroke (3), heart disease (4), and atherosclerosis (5, 6). The potential cellular targets for peroxynitrite cytotoxicity include the antioxidants ascorbate, ␣-tocopherol, and uric acid (7-10), protein and non-protein sulfhydryls (11), DNA (12), and membrane phospholipids (13). Decomposition of peroxynitrite is complex (14, 15). The anion is rather stable in alkaline solutions but decomposes rapidly (t 1/2 ϭ 1 s at pH 7.4, 37°C) upon protonation to peroxynitrous acid (ONOOH) (pK a ϭ 6.8) (16). Two pathways of ONOOH decomposition have been proposed. Some studies have argued that ONOOH is cleaved homolytically to generate hydroxyl and NO 2 radicals. This hypothesis is based on the sensitivity to hydroxyl radical scavengers of certain peroxynitrite-induced reactions, including the formation of malondialdehyde from deoxyribose and the hydroxylation on the benzene ring of sodium benzoate, phenylalanine, and tyrosine (16, 17). Studies on decomposition of peroxynitrite by electron paramagnetic resonance spectroscopy with the spin traps 5,5-dime...

The properties of neuronal nitric oxide synthase containing one tetrahydrobiopterin (BH4) per dimer [nNOS(BH4+)] were compared to those of the BH4-free enzyme [nNOS(BH4-)]. The stimulation by BH4 of the formation of L-citrulline at the expense of H2O2 production unambiguously demonstrated that BH4 is essential in coupling reductive oxygen activation to Arg oxidation. The clear difference between the Stokes radii of nNOS(BH4-) and nNOS(BH4+) indicates that the introduction of one BH4 per dimer significantly changes the enzyme structure. Whereas the heme in nNOS(BH4+) was primarily high-spin, nNOS(BH4-) contained mainly low-spin heme. This was slowly converted into the high-spin form with Arg and/or BH4, with a rate that was independent of the concentration of either compound. Dithiothreitol inhibited the Arg/BH4-induced spin conversion by stabilizing low-spin heme. Formation of high-spin heme, with rates varying from 0.04 to 0.4 min-1, always correlated to an equally fast increase in activity. Radioligand binding studies showed the rapid association (within 20 s) of BH4 to nNOS(BH4-), but not to nNOS(BH4+), after preincubation with Arg. Complete and monophasic dissociation of radioligand occurred in the presence of excess unlabeled BH4, demonstrating the exchangeability of high-affinity bound BH4. Studies of the association of NG-nitro-L-arginine (L-NNA) to nNOS(BH4+) revealed that excess BH4 increased the amount of bound L-NNA 2-fold. Most of the binding data are explained by a model in which nNOS dimers accommodate two identical BH4- and Arg/L-NNA-binding sites, with cooperativity between Arg- and BH4-binding and anticooperativity between the BH4-binding sites.

The reaction of reduced NO synthase (NOS) with molecular oxygen was studied at ؊30°C. In the absence of substrate, the complex formed between ferrous NOS and O 2 was sufficiently long lived for a precise spectroscopic characterization. This complex displayed similar spectral characteristics as the oxyferrous complex of cytochrome P450 ( max ‫؍‬ 416.5 nm). It then decomposed to the ferric state. The oxidation of the flavin components was much slower and could be observed only at temperatures higher than ؊20°C.

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