%0 Journal Article %A Guerra, Damian %A Ballard, Keith %A Truebridge, Ian %A Vierling, Elizabeth %D 2016 %T S‑Nitrosation of Conserved Cysteines Modulates Activity and Stability of S‑Nitrosoglutathione Reductase (GSNOR) %U https://acs.figshare.com/articles/journal_contribution/S_Nitrosation_of_Conserved_Cysteines_Modulates_Activity_and_Stability_of_i_S_i_Nitrosoglutathione_Reductase_GSNOR_/3187093 %R 10.1021/acs.biochem.5b01373.s001 %2 https://acs.figshare.com/ndownloader/files/4975393 %K GSNOR %K GSNO accumulation %K signal %K Conserved Cysteines Modulates Activity %K mass spectrometry %K nitroso donors %K DTT %K coenzyme binding pockets %K nitrosation %X The free radical nitric oxide (NO) regulates diverse physiological processes from vasodilation in humans to gas exchange in plants. S-Nitrosoglutathione (GSNO) is considered a principal nitroso reservoir due to its chemical stability. GSNO accumulation is attenuated by GSNO reductase (GSNOR), a cysteine-rich cytosolic enzyme. Regulation of protein nitrosation is not well understood since NO-dependent events proceed without discernible changes in GSNOR expression. Because GSNORs contain evolutionarily conserved cysteines that could serve as nitrosation sites, we examined the effects of treating plant (Arabidopsis thaliana), mammalian (human), and yeast (Saccharomyces cerevisiae) GSNORs with nitrosating agents in vitro. Enzyme activity was sensitive to nitroso donors, whereas the reducing agent dithiothreitol (DTT) restored activity, suggesting that catalytic impairment was due to S-nitrosation. Protein nitrosation was confirmed by mass spectrometry, by which mono-, di-, and trinitrosation were observed, and these signals were sensitive to DTT. GSNOR mutants in specific non-zinc-coordinating cysteines were less sensitive to catalytic inhibition by nitroso donors and exhibited reduced nitrosation signals by mass spectrometry. Nitrosation also coincided with decreased tryptophan fluorescence, increased thermal aggregation propensity, and increased polydispersityproperties reflected by differential solvent accessibility of amino acids important for dimerization and the shape of the substrate and coenzyme binding pockets as assessed by hydrogen–deuterium exchange mass spectrometry. Collectively, these data suggest a mechanism for NO signal transduction in which GSNOR nitrosation and inhibition transiently permit GSNO accumulation. %I ACS Publications