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

Dragana Nikitovic,Arne Holmgren

doi:10.1074/jbc.271.32.19180

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 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.

Highlights

In physiological systems NO1 has many functions in redox signaling [1,2,3,4]
Glutathione reductase from yeast or rat liver showed no activity with GSH to S-nitrosoglutathione (GSNO) in agreement with the result of others [17] and the preparation of thioredoxin reductase (TR) used was devoid of such activity
Our results demonstrate that GSNO is reduced by either human or calf thymus thioredoxin reductase or by thioredoxin

Summary

Introduction

In physiological systems NO1 has many functions in redox signaling [1,2,3,4]. Among targets of NO, protein thiols are S-nitrosylated [5,6,7]. 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]. NADPH will reduce an active site disulfide in thioredoxin reductase (TR) which reduces oxidized thioredoxin (Trx-S2) to Trx-(SH) which is reoxidized in a direct reaction with a disulfide (Reactions 1–3). Since the disulfides of insulin efficiently oxidize Trx-(SH) (Reaction 3) [15], this protein is used as a classical substrate to test the activity of the Trx system [15]. The direct reaction between Trx-(SH) and insulin (K2 Ͼ5 ϫ 104 MϪ1sϪ1) is more than 104 times faster than the equivalent reaction with the well known dithiothreitol (DTT) molecule [13]. The well characterized Escherichia coli TR (Mr70,000) is highly specific for the homologous Trx and some related prokaryotic thioredoxins [13,14,15]

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Conclusion