REACTIVITY OF NITROGEN MONOXIDE SPECIES WITH NADH - IMPLICATIONS FOR NITRIC OXIDE-DEPENDENT POSTTRANSLATIONAL PROTEIN MODIFICATION

Nitric oxide (NO .) and NO . donors incite NAD- [i.e., mono(ADP-ribosy lation)] and NADH-dependent posttranslational protein modifications by an as yet unknown mechanism. A route of pyridine nucleotide-dependent , NO .-stimulated protein modification has recently been hypothesized [S. Dimmeler, and B. Brune, (1992) Eur. J. Biochem. 210, 305-310; J.S. Stamler (1994) Cell 78, 931-936], An essential feature of this propos ed mechanism is NADH nitrosation, for a nitroso-NADH adduct is conside red to be a key reactant in the generation of pyridine nucleotide-modi fied protein. To evaluate at the molecular level the ability of NADH t o act as a nitrosation substrate, the potential effects of NO ., the n itrosothiols S-nitrosoglutathione and S-nitrosocysteine, the nitrosati ng agent tert-butyl-nitrite, and the NO . metabolite peroxynitrite on the molecular and functional (i.e., hydride-transfer) properties of NA DH have been directly assessed at physiological pH, Exposure of NADH t o NO . or nitrosothiol altered neither the hydride-transfer capability of the pyridine nucleotide nor its ultraviolet spectrum in ways sugge stive of NADH nitrosation. As determined by MMR spectroscopy, NADH was refractory to the well-recognized nitrosating agent tert-butyl nitrit e, Consequently, it appears that NADH is an unfavorable substrate for nitrosation under physiological conditions, These data are inconsisten t with the proposal that NO . or a NO .-derived nitrosating agent inte racts with NADH to generate the nitroso-NADH hypothesized to be essent ial to NO .-stimulated, pyridine nucleotide-dependent protein modifica tion, Peroxynitrite, a possible source of nitrosating compounds, readi ly oxidized NADH to NAD, but demonstrated no potential to form a nitro so-NADH adduct, The facility with which NADH is oxidized to NAD has im plications for peroxynitrite-mediated tissue damage. (C) 1997 Academic Press