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 nucleotidedependent, NO•-stimulated protein modification has recently been hypothesized [S. Dimmeler, and B. Brune, (1992)Eur. J. Biochem.210, 305–310; J. S. Stamler (1994)Cell78, 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 nitrosothiolsS-nitrosoglutathione and S-nitrosocysteine, the nitrosating agenttert-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 NMR spectroscopy, NADH was refractory to the well-recognized nitrosating agenttert-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.