HNO induces DNA deletions in the yeast S. cerevisiae.

HNO is genotoxic but its mechanism is not well understood. There are many possible mechanisms by which HNO can attack DNA. Since HNO is electrophilic, it may react with exocyclic amine groups on DNA bases and through a series of subsequent reactions form a deaminated product. Alternatively, HNO may induce radical chemistry through O(2)-dependent (or possibly O(2)-independent) chemistry. In cell free systems, experiments have shown that HNO does react with DNA, resulting in base oxidation and strand cleavage. In this study, we used a whole-cell system in the yeast Saccharomyces cerevisiae to study the mechanism of HNO induced DNA damage with Angeli's salt as HNO donor. The yeast DEL assay provided a measure of intrachromosomal recombination leading to DNA deletions. We also examined interchromosomal recombination leading to genomic rearrangements and used the canavanine (CAN) assay to study induction of forward point mutations. HNO was a potent inducer of DNA deletions and recombination but it was negative for induction of point mutations. This suggests that HNO causes DNA strand breaks rather than base damage. Genotoxicity was observed under aerobic and anaerobic conditions and NAC protected against HNO induced DNA deletions. Since HNO is genotoxic under anaerobic conditions, NAC probably protected against radicals generated by HNO independent of oxygen.

Inhibition of yeast glycolysis by nitroxyl (HNO): mechanism of HNO toxicity and implications to HNO biology.

Nitroxyl (HNO) was found to inhibit glycolysis in the yeast Saccharomyces cerevisiae. The toxicity of HNO in yeast positively correlated with the dependence of yeast on glycolysis for cellular energy. HNO was found to potently inhibit the crucial glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH), an effect which is likely to be responsible for the observed inhibition of glycolysis in whole cell preparations. It is proposed that GAPDH inhibition occurs through reaction of HNO with the active site thiolate residue of GAPDH. Significantly, levels of HNO that inhibit GAPDH do not alter the levels or redox status of intracellular glutathione (GSH), indicating that HNO has thiol selectivity. The ability of HNO to inhibit GAPDH in an intracellular environment that contains relatively large concentrations of GSH is an important aspect of HNO pharmacology and possibly, physiology.

Nitroxyl-mediated disruption of thiol proteins: inhibition of the yeast transcription factor Ace1.

Among the biologically and pharmacologically relevant nitrogen oxides, nitroxyl (HNO) remains one of the most poorly studied and least understood. Several previous reports indicate that thiols may be a primary target for the biological actions of HNO. However, the intimate details of the chemical interaction of HNO with biological thiols remain unestablished. Due to their ability to grow under a variety of conditions, the yeast Saccharomyces cerevisiae represents a unique and useful model system for examining the chemistry of HNO with thiol proteins in a whole-cell preparation. Herein, we have examined the effect of HNO on the thiol-containing, metal-responsive, yeast transcription factor Ace1 under a variety of cellular conditions as a means of delineating the chemistry of HNO interactions with this representative thiol protein. Using a reporter gene system, we find that HNO efficiently inhibits copper-dependent Ace1 activity. Moreover, this inhibition appears to be a result of a direct interaction between Ace1 thiols and HNO and not a result of any chemistry associated with HNO-derived species. Thus, this report indicates that thiol proteins can be a primary target of HNO biochemistry and that HNO-mediated thiol modification is likely due to a direct reaction of HNO.