Reactivity of nitrogen monoxide species with NADH: implications for nitric oxide-dependent posttranslational protein...

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 NMR 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.