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The reaction kinetics of nitric oxide autoxidation in aerobic solutions were investigated by direct observation of the nitrite ion product and by trapping the strongly oxidizing and nitrosating intermediates formed in this reaction. The rate behavior observed for nitrite formation [rate = k3[O2][NO]2, k3 = (6 +/- 1.5) x 10(6) M-2 s-1 at 22 degrees C] was the same as found for oxidation of Fe(CN)6(4-) and of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and as for the nitrosation of sulfanilamide. There was a slight decrease in k3 to (3.5 +/- 0.7) x 10(6) M-2 s-1 at 37 degrees C. The second-order dependency for NO was observed at NO concentrations as low as 3 microM. The results of the competitive kinetics studies suggest that the key oxidizing intermediates, species which are both strong oxidants and nitrosating agents, are not one of those commonly proposed (NO2, N2O3, NO+, or O2NO-) but are one or more as yet uncharacterized NOx species.

The critical regulatory function of nitric oxide (NO) in many physiologic processes is well established. However, in an aerobic aqueous environment NO is known to generate one or more reactive and potentially toxic nitrogen oxide (NOx) metabolites. This has led to the speculation that mechanisms must exist in vivo by which these reactive intermediates are detoxified, although the nature of these mechanisms has yet to be elucidated. This report demonstrates that among the primary bioorganic products of the reaction of cellular constituents with the intermediates of the NO/O2 reaction are S-nitrosothiol (S-NO) adducts. Anaerobic solutions of NO are not capable of nitrosating cysteine or glutathione, while S-NO adducts of these amino acids are readily formed in the presence of O2 and NO. Investigation of the kinetics for the formation of these S-NO adducts has revealed a rate equation of d[RSNO]/dt = kSNO[NO]2[O2], where kSNO = (6 +/- 2) x 10(6) M-2S-1, a value identical to that for the formation of reactive intermediates in the autoxidation of NO. Competition studies performed with a variety of amino acids, glutathione, and azide have shown that cysteine residues have an affinity for the NOx species that is 3 orders of magnitude greater than that of the nonsulfhydryl amino acids, and > 10(6) times greater than that of the exocyclic amino groups of DNA bases. The dipeptide alanyltyrosine reacts with the intermediates of the NO/O2 reaction with an affinity 150 times less than that of the sulfhydryl-containing compounds. Furthermore, Chinese hamster V79 lung fibroblasts depleted of glutathione display enhanced cytotoxicity on exposure to NO.(ABSTRACT TRUNCATED AT 250 WORDS)

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