S-nitrosoglutathione is cleaved by the thioredoxin system with liberation of glutathione and redox regulating nitric oxide.
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 Km value of 60 microM and a Kcat of 0.6 x 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-S2 by GSNO with a rate constant of 760 M-1s-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. 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.. 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.
Nikitovic
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D
Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden.
Holmgren
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A
D000818:Animals; D002417:Cattle; D004926:Escherichia coli / Q000201:enzymology; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D009211:Myoglobin / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D009603:Nitroso Compounds / Q000378:metabolism*; D010084:Oxidation-Reduction; D026422:S-Nitrosoglutathione; D013379:Substrate Specificity; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism; D013879:Thioredoxins / Q000037:antagonists & inhibitors / Q000378:metabolism*
D009211:Myoglobin; D009603:Nitroso Compounds; D009569:Nitric Oxide; D013879:Thioredoxins; D026422:S-Nitrosoglutathione; D013880:Thioredoxin-Disulfide Reductase; D005978:Glutathione