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J. Biol. Chem., Vol. 282, Issue 19, 14428-14436, May 11, 2007

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Oxidation and S-Nitrosylation of Cysteines in Human Cytosolic and Mitochondrial Glutaredoxins

EFFECTS ON STRUCTURE AND ACTIVITY^*

Seyed Isaac Hashemy¹², Catrine Johansson¹³, Carsten Berndt⁴, Christopher Horst Lillig⁵, and Arne Holmgren⁶

From the Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden and Department of Clinical Cytobiology and Cytopathology, Philipps University Marburg, DE-35037 Marburg, Germany

Glutathione (GSH) is the major intracellular thiol present in 1–10-mM concentrations in human cells. However, the redox potential of the 2GSH/GSSG (glutathione disulfide) couple in cells varies in association with proliferation, differentiation, or apoptosis from -260 mV to -200 or -170 mV. Hydrogen peroxide is transiently produced as second messenger in receptor-mediated growth factor signaling. To understand oxidation mechanisms by GSSG or nitric oxide-related nitrosylation we studied effects on glutaredoxins (Grx), which catalyze GSH-dependent thiol-disulfide redox reactions, particularly reversible glutathionylation of protein sulfhydryl groups. Human Grx1 and Grx2 contain Cys-Pro-Tyr-Cys and Cys-Ser-Tyr-Cys active sites and have three and two additional structural Cys residues, respectively. We analyzed the redox state and disulfide pairing of Cys residues upon GSSG oxidation and S-nitrosylation. Cytosolic/nuclear Grx1 was partly inactivated by both S-nitrosylation and oxidation. Inhibition by nitrosylation was reversible under anaerobic conditions; aerobically it was stronger and irreversible, indicating inactivation by nitration. Oxidation of Grx1 induced a complex pattern of disulfide-bonded dimers and oligomers formed between Cys-8 and either Cys-79 or Cys-83. In addition, an intramolecular disulfide between Cys-79 and Cys-83 was identified, predicted to have a profound effect on the three-dimensional structure. In contrast, mitochondrial Grx2 retains activity upon oxidation, did not form disulfide-bonded dimers or oligomers, and could not be S-nitrosylated. The dimeric iron sulfur cluster-coordinating inactive form of Grx2 dissociated upon nitrosylation, leading to activation of the protein. The striking differences between Grx1 and Grx2 reflect their diverse regulatory functions in vivo and also adaptation to different subcellular localization.

Received for publication, January 31, 2007 , and in revised form, March 6, 2007.

^* This work was supported in part by grants from the Karolinska Institute, the Swedish Cancer Society, the Swedish Research Council, the Swedish Society for Medical Research, and the Wenner-Gren Foundation. 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 Both authors contributed equally to this work.

² Supported by a scholarship from the ministry of Health I. R. Iran.

³ Present address: Biotechnology, The Structural Genomics Consortium, University of Oxford, Botnar Research Centre, Oxford OX3 7LD, UK.

⁴ Supported by a scholarship from the Deutsche Forschungsgemeinschaft.

⁵ Supported by a scholarship from the Swedish Society for Medical Research.

⁶ To whom correspondence should be addressed. Tel.: 46-8-524-87686; Fax: 46-8-7284716; E-mail: arne.holmgren{at}ki.se.

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