S-Nitrosoglutathione Is Cleaved by the Thioredoxin System with Liberation of Glutathione and Redox Regulating Nitric Oxide

Abstract

In activated human neutrophils a burst of nitric oxide (NO) converts intracellular GSH to S-nitrosoglutathione (GSNO) which is subsequently cleaved to restore GSH by an unknown mechanism. We discovered that GSNO is an NADPH oxidizing substrate for human or calf thymus thioredoxin reductase (TR) with an apparent K m value of 60 M and a K cat of 0.6 ؋ s ؊1 . Addition of human thioredoxin (Trx) stimulated the initial NADPH oxidation rate severalfold but was accompanied by progressive inactivation of TR. Escherichia coli TR lacked activity with GSNO, but with E. coli Trx present, GSNO was reduced without inhibition of the enzyme. Chemically reduced E. coli Trx-(SH) 2 was oxidized to Trx-S 2 by GSNO with a rate constant of 760 M ؊1 s ؊1 (7-fold faster than by GSSG) as measured by tryptophan fluorescence. Analysis of this reaction in the presence of oxymyoglobin revealed quantitative formation of metmyoglobin indicative of NO ⅐ release. Analysis of GSNO reduction demonstrated that oxidation of NADPH produced a stoichiometric amount of free GSH. These results demonstrate a homolytic cleavage mechanism of GSNO, giving rise to GSH and NO ⅐ . GSNO efficiently inhibited the protein disulfide reductase activity of the complete human or calf thymus thioredoxin systems. Our results demonstrate enzymatic cleavage of GSNO by TR or Trx and suggest novel mechanisms for redox signaling.In physiological systems NO 1 has many functions in redox signaling (1-4). Among targets of NO, protein thiols are specifically S-nitrosylated (5-7). Thus, nitrosylation of a critical Cys-residue in p21 ras activates this G protein (8). In cells glutathione readily reacts with activated NO intermediates, forming the stable adduct S-nitrosoglutathione (GSNO) as shown in activated human neutrophils (9, 10). The observed time-dependent recovery of GSH in the cells (10, 11) could not be accounted for by spontaneous degradation of GSNO. The mechanism of cleavage is thus unknown.Thioredoxin is a 12-kDa protein with a redox active disulfide in the conserved active site sequence -Cys-Gly-Pro-Cys-located on a protrusion in its three-dimensional structure (12). Reduced thioredoxin (Trx-(SH) 2 ) operates together with thioredoxin reductase and NADPH (the thioredoxin system) as a general protein disulfide reductase (13,14). NADPH will reduce an active site disulfide in thioredoxin reductase (TR) which then reduces oxidized thioredoxin (Trx-S 2 ) to Trx-(SH) 2 which is reoxidized in a direct reaction with a disulfide (Reactions 1-3). ) is more than 10 4 times faster than the equivalent reaction with the well known dithiothreitol (DTT) molecule (13). Mammalian thioredoxin reductases (M r 116,000) show an intriguing substrate specificity, reducing not only thioredoxins from many species but also 5,5Ј-dithiobis-(2-nitrobenzoicacid) (DTNB), selenodiglutathione, vitamin K, or alloxan (13-15). In contrast, the well characterized Escherichia coli TR (M r 70,000) is highly specific for the homologous Trx and some related prokaryotic thioredoxins (13-15).The aim of the p...

TNFα and oxLDL Reduce Protein S-Nitrosylation in Endothelial Cells

“…A possible explanation of why GSNO did not accumulate in the cells could be a rapid metabolization. In support of this hypothesis, the thioredoxin system was shown to rapidly cleave NO out of GSNO in a cell-free system (29). In addition, a glutathionedependent formaldehyde dehydrogenase was identified in eukaryotes, which specifically denitrosylates GSNO (21).…”

Glutathione disulfide and S‐nitrosoglutathione detoxification by Mycobacteriumtuberculosis thioredoxin system

“…Denitrosylation of GSNO by human thioredoxins has been studied by both the Holmgren and Stoyanovsky laboratories. Holmgren's group has reported a homolytic breakdown of GSNO generating GSH and NO [27], while Stoyanovsky has proposed a heterolytic breakdown of GSNO producing GSH and nitroxyl (HNO) [28]. However both groups suggest that other cellular nitrosylated proteins may serve as substrates for the thioredoxin system.…”

Redox-based regulation of signal transduction: Principles, pitfalls, and promises

“…S-nitrosylation of cysteine 72 has been recently implicated in transnitrosation of caspases, but did not seem to affect Trx activity [111,112]. Interestingly Trx may also impact S-nitrosothiol homeostasis via its ability to cleave GSNO and protein SNO [60,113]. These collective observations point to important cross regulation of the various redox systems which may have impact on steady state levels of various cysteine oxidation states controlled by these systems.…”