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-ribosylation)] 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 proposed mechanism is NADH nitrosation, for a nitroso-NADH adduct is considered to be a key reactant in the generation of pyridine nucleotide-modified protein. To evaluate at the molecular level the ability of NADH to act as a nitrosation substrate, the potential effects of NO ., the nitrosothiols S-nitrosoglutathione and S-nitrosocysteine, the nitrosating agent tert-butyl-nitrite, and the NO . metabolite peroxynitrite on the molecular and functional (i.e., hydride-transfer) properties of NADH have been directly assessed at physiological pH, Exposure of NADH to NO . or nitrosothiol altered neither the hydride-transfer capability of the pyridine nucleotide nor its ultraviolet spectrum in ways suggestive of NADH nitrosation. As determined by MMR spectroscopy, NADH was refractory to the well-recognized nitrosating agent tert-butyl nitrite, Consequently, it appears that NADH is an unfavorable substrate for nitrosation under physiological conditions, These data are inconsistent with the proposal that NO . or a NO .-derived nitrosating agent interacts with NADH to generate the nitroso-NADH hypothesized to be essential to NO .-stimulated, pyridine nucleotide-dependent protein modification, Peroxynitrite, a possible source of nitrosating compounds, readily oxidized NADH to NAD, but demonstrated no potential to form a nitroso-NADH adduct, The facility with which NADH is oxidized to NAD has implications for peroxynitrite-mediated tissue damage. (C) 1997 Academic Press