Reduction of S‐nitrosoglutathione by human alcohol dehydrogenase 3 is an irreversible reaction as analysed by electrospray mass spectrometry

Abstract

Human alcohol dehydrogenase 3/glutathione-dependent formaldehyde dehydrogenase was shown to rapidly and irreversibly catalyse the reductive breakdown of S-nitrosoglutathione. The steady-state kinetics of S-nitrosoglutathione reduction was studied for the wild-type and two mutated forms of human alcohol dehydrogenase 3, mutations that have previously been shown to affect the oxidative efficiency for the substrate S-hydroxymethylglutathione. Wild-type enzyme readily reduces S-nitrosoglutathione with a k cat /K m approximately twice the k cat /K m for S-hydroxymethylglutathione oxidation, resulting in the highest catalytic efficiency yet identified for a human alcohol dehydrogenase. In a similar manner as for S-hydroxymethylglutathione oxidation, the catalytic efficiency of S-nitrosoglutathione reduction was significantly decreased by replacement of Arg115 by Ser or Lys, supporting similar substrate binding. NADH was by far a better coenzyme than NADPH, something that previously has been suggested to prevent reductive reactions catalysed by alcohol dehydrogenases through the low cytolsolic NADH/NAD + ratio. However, the major products of S-nitrosoglutathione reduction were identified by electrospray tandem mass spectrometry as glutathione sulfinamide and oxidized glutathione neither of which, in their purified form, served as substrate or inhibitor for the enzyme. Hence, the reaction products are not substrates for alcohol dehydrogenase 3 and the overall reaction is therefore irreversible. We propose that alcohol dehydrogenase 3 catalysed S-nitrosoglutathione reduction is of physiological relevance in the metabolism of NO in humans.

A Glutathione-Dependent Detoxification System Is Required for Formaldehyde Resistance and Optimal Survival ofNeisseria meningitidisin Biofilms

“…It has been suggested that the interplay between the metabolism of GSH and its derivatives, including GSNO and HMGSH, could have a role in regulation of NO signaling via S-nitrosylation of proteins (41). In fact, in mammals, yeast, and E. coli, AdhC is known to catalyze the NADH-dependent reduction of GSNO (30), and it has been proposed that this enzyme protects against nitrosative stress (17,30,42). Interestingly, EstD is not generally considered to play a role in the removal of the products of GSNO reduction (41).…”

The Specificity of Nitroxyl Chemistry Is Unique Among Nitrogen Oxides in Biological Systems

“…Recent experimental and computational analyses of these interactions have clarified mechanistic details (60,148,149). High-performance liquid chromatography detection of sulfinamide has also been offered as an indicator of HNO production (34).…”

Identification of Nitroxyl-induced Modifications in Human Platelet Proteins Using a Novel Mass Spectrometric Detection Method

“…As well, the sulfinamide modification imparts a specific mass change to cysteines making sulfinamide analysis, and indirectly HNO analysis, very amenable to investigation by mass spectrometry. The sulfinamide modification has been observed in MS spectra (28), and in a recent mass spectrometric analysis, Shen and English (29) attributed a mass shift of 65 Da on prominent y-ions upon low energy CID to the elimination of the sulfinamide moiety from the molecule in their mass spectrometric comparison of nitroxyl products formed with free and protein-based cysteines. Here we investigate this mass shift and the formation of a previously unstudied neutral loss to determine an efficient method for the identification of the sulfinamide modification and demonstrate its utility on a sample generated by treatment of live platelets immediately post-isolation, that is ex vivo, with HNO.…”