Oxidation and nitrosation in the nitrogen monoxide/superoxide system

Area of Science:

• Biochemistry and Molecular Biology
• Enzymology
• Oxidative Stress Research

Background:

• Alcohol dehydrogenase (ADH) possesses a zinc dithiolate active site susceptible to cellular oxidants.
• Previous studies by McCord and co-workers highlighted the potential reactivity of this site.

Purpose of the Study:

• To investigate the inactivation mechanism of ADH by cellular oxidants, specifically focusing on the zinc dithiolate active site.
• To elucidate the roles of nitrogen monoxide (NO) and superoxide (O2) in ADH activity modulation.
• To explore the protective effects of excess NO against oxidative damage.

Main Methods:

• Enzyme activity assays to measure ADH inactivation kinetics.
• Generation of reactive oxygen and nitrogen species (e.g., peroxynitrite, NO, O2) using various chemical systems (e.g., xanthine oxidase, 3-morpholino sydnonimine).
• Quantification of zinc release and disulfide formation.
• Identification of zinc coordination sites (Cys2His and Cys4) using chelating agents like PAR.

Main Results:

• Equimolar generation of NO and O2 led to rapid ADH inactivation via peroxynitrite formation, with significantly lower IC50 values compared to H2O2 or NO alone.
• Superoxide generated by xanthine oxidase was also effective in ADH inactivation.
• Inactivation correlated with zinc release and disulfide formation, primarily from the zinc Cys2His center.
• Excess NO abolished peroxynitrite-mediated inactivation, suggesting the formation of nitrosating species that block oxidative damage.

Conclusions:

• Low fluxes of NO and O2 in cellular systems can generate peroxynitrite, effectively oxidizing the zinc thiolate sites of ADH.
• The nucleophilic nature of the thiolate group facilitates these oxidative modifications.
• Excess NO exhibits antioxidant properties by blocking peroxynitrite actions, potentially through S-nitrosation mechanisms.