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J. Biol. Chem., Vol. 283, Issue 34, 22890-22894, August 22, 2008

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Identification of Intact Protein Thiosulfinate Intermediate in the Reduction of Cysteine Sulfinic Acid in Peroxiredoxin by Human Sulfiredoxin^*

Thomas J. Jönsson, Allen W. Tsang, W. Todd Lowther¹, and Cristina M. Furdui²

From the Center for Structural Biology and Department of Biochemistry and the Section on Molecular Medicine, Department of Internal Medicine, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina 27157

The reversible oxidation of the active site cysteine in typical 2-Cys peroxiredoxins (Prx) to sulfinic acid during oxidative stress plays an important role in peroxide-mediated cell signaling. The catalytic retroreduction of by sulfiredoxin (Srx) has been proposed to proceed through two novel reaction intermediates, a sulfinic phosphoryl ester and protein-based thiosulfinate. Two scenarios for the repair mechanism have been suggested that differ in the second step of the reaction. The attack of Srx or GSH on the intermediate would result in either the formation of Prx-Cys-S(=O)–S-Cys-Srx or the formation of Prx-Cys-S(=O)–S-G thiosulfinates, respectively. To elucidate the mechanism of Prx repair, we monitored the reduction of human using rapid chemical quench methodology and electrospray ionization time-of-flight mass spectrometry. An ¹⁸O exchange study revealed that the Prx sulfinic acid phosphoryl ester is rapidly formed and hydrolyzed (k = 0.35 min^–1). Furthermore, we observed the exclusive formation of a thiosulfinate linkage between Prx and Srx (k = 1.4 min^–1) that collapses to the disulfide-bonded Srx-Prx species (k = 0.14 min^–1). Thus, the kinetic and chemical competences of the first two steps in the Srx reaction have been demonstrated. It is clear, however, that GSH may influence thiosulfinate formation and that GSH and Srx may play additional roles in the resolution of the thiosulfinate intermediate.

Received for publication, June 19, 2008 , and in revised form, June 30, 2008.

Note Added in Proof—Recent studies of the yeast Srx/Prx system have also led to the identification of the Prx-Cys-S(=O)-S-Cys-Srx thiosulfinate catalytic intermediate (25).

^* This work was supported, in whole or in part, by National Institutes of Health Grant R01GM072866 (to W. T. L.). This work was also supported by American Heart Association Postdoctoral Fellowship 0725399U (to T. J. J.) and American Heart Association Grant SDG 0730069N (to C. M. F.). 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 To whom correspondence may be addressed: Center for Structural Biology and Dept. of Biochemistry, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157. Tel.: 336-716-7230; Fax: 336-777-3242; E-mail: tlowther{at}wfubmc.edu. ² To whom correspondence may be addressed: Section on Molecular Medicine, Dept. of Internal Medicine, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157. Tel.: 336-716-2697; Fax: 336-716-1214; E-mail: cfurdui{at}wfubmc.edu.

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