%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 polydispersityproperties
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