New insights into the S-nitrosothiol–ascorbate reaction. The formation of nitroxyl

Abstract

Although the ascorbate-dependent reduction of S-nitrosothiol to the corresponding thiol function is frequently used for analyzing S-nitrosocysteinyl residues in proteins, it proceeds with low yields. Our re-investigation of the ascorbate–S-nitrosothiol reaction demonstrates now the intermediacy of nitroxyl (HNO/³NO⁻) that is highly effective in oxidizing thiols. The occurrence of the HNO reporter molecule, i.e., N₂O, during and after reaction was unequivocally demonstrated by ¹⁵N NMR. The yield of HNO from the S-nitrosoglutathione–ascorbate reaction was determined with the aid of a Mn(III)-complex to 60%, a value that was significantly higher than the one of nitric oxide formation (48%) at physiological pH. The same HNO yield was observed with a S-nitrosothiol bound to a protein (i.e., S-nitroso-papain). With the known chemistry of nitroxyl, it was possible to optimize the experimental conditions so that the GSNO–ascorbate reaction yielded stoichiometric amounts of glutathione after a reaction period of 1 min.