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J. Biol. Chem., Vol. 283, Issue 32, 21890-21898, August 8, 2008

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Regulation of the Catalytic Activity and Structure of Human Thioredoxin 1 via Oxidation and S-Nitrosylation of Cysteine Residues^*

From the Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden

The mammalian cytosolic/nuclear thioredoxin system, comprising thioredoxin (Trx), selenoenzyme thioredoxin reductase (TrxR), and NADPH, is the major protein-disulfide reductase of the cell and has numerous functions. The active site of reduced Trx comprises Cys³²-Gly-Pro-Cys³⁵ thiols that catalyze target disulfide reduction, generating a disulfide. Human Trx1 has also three structural Cys residues in positions 62, 69, and 73 that upon diamide oxidation induce a second Cys⁶²–Cys⁶⁹ disulfide as well as dimers and multimers. We have discovered that after incubation with H₂O₂ only monomeric two-disulfide molecules are generated, and they are inactive but able to regain full activity in an autocatalytic process in the presence of NADPH and TrxR. There are conflicting results regarding the effects of S-nitrosylation on Trx antioxidant functions and which residues are involved. We found that S-nitrosoglutathione-mediated S-nitrosylation at physiological pH is critically dependent on the redox state of Trx. Starting from fully reduced human Trx, both Cys⁶⁹ and Cys⁷³ were nitrosylated, and the active site formed a disulfide; the nitrosylated Trx was not a substrate for TrxR but regained activity after a lag phase consistent with autoactivation. Treatment of a two-disulfide form of Trx1 with S-nitrosoglutathione resulted in nitrosylation of Cys⁷³, which can act as a trans-nitrosylating agent as observed by others to control caspase 3 activity (Mitchell, D. A., and Marletta, M. A. (2005) Nat. Chem. Biol. 1, 154–158). The reversible inhibition of human Trx1 activity by H₂O₂ and NO donors is suggested to act in cell signaling via temporal control of reduction for the transmission of oxidative and/or nitrosative signals in thiol redox control.

Received for publication, February 8, 2008 , and in revised form, May 26, 2008.

^* This work was supported by Swedish Cancer Society Grant 961, Swedish Research Council of Medicine Grant 13x-3529, and the K. A. Wallenberg Foundation and the Karolinska Institutet. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by a scholarship from the Ministry of Health and Medical Education of Iran. Present address: Dept. of Biochemistry and Nutrition, Mashhad University of Medical Sciences, Mashhad, Iran.

² To whom correspondence should be addressed: Medical Nobel Inst. for Biochemistry, Dept. of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden. Tel.: 46-8-52487686; Fax: 46-8-7284716; E-mail: arne.holmgren{at}ki.se.

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