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J. Biol. Chem., Vol. 282, Issue 43, 31429-31436, October 26, 2007

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Molecular Mechanism of Oxidative Stress Perception by the Orp1 Protein^*

From the Department of Environmental Toxicology, University of California, Davis, California 95616

In this study we investigated the molecular mechanism by which the Orp1 (Gpx3) protein in Saccharomyces cerevisiae senses and reacts with hydrogen peroxide. Upon exposure to H₂O₂ Orp1^Cys36 forms a disulfide-bonded complex with the C-terminal domain of the Yap1 protein (Yap1-cCRD). We used 4-nitrobenzo-2-oxa-1,3-diazole to identify a cysteine sulfenic acid (Cys-SOH) modification that forms on Cys³⁶ of Orp1^Cys36 upon exposure to H₂O₂. Under similar conditions, neither Cys⁸² of Orp1^Cys82 nor Cys⁵⁹⁸ of Yap1 forms Cys-SOH. A homology-based molecular model of Orp1 suggests that the structure of the active site of Orp1 is similar to that found in mammalian selenocysteine glutathione peroxidases. Proposed active site residues Gln⁷⁰ and Trp¹²⁵ form a catalytic triad with Cys³⁶ in the Orp1 molecular model. The remainder of the active site pocket is formed by Phe³⁸, Asn¹²⁶, and Phe¹²⁷, which are evolutionarily conserved residues. We made Q70A and W125A mutants and tested the ability of these mutants to form Cys-SOH in response to H₂O₂. Both mutants were unable to form Cys-SOH and did not form a H₂O₂-inducible disulfide-bonded complex with Yap1-cCRD. The pK_a of Cys³⁶ was determined to be 5.1, which is 3.2 pH units lower than that of a free cysteine (8.3). In contrast, Orp1 Cys⁸² (the resolving cysteine) has a pK_a value of 8.3. The pK_a of Cys³⁶ in the Q70A and W125A mutants is also 8.3, demonstrating the importance of these residues in modulating the nucleophilic character of Cys³⁶. Finally, we show that S. cerevisiae strains with ORP1 Q70A and W125A mutations are less tolerant to H₂O₂ than those containing wild-type ORP1. The results of our study suggest that attempts to identify novel redox-regulated proteins and signal transduction pathways should focus on characterization of low pK_a cysteines.

Received for publication, July 20, 2007 , and in revised form, August 21, 2007.

^* 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 should be addressed: Dept. of Environmental Toxicology, University of California, Davis, One Shields Ave., Davis, CA 95616. Tel.: 530-754-2271; Fax: 530-752-3394; E-mail: mjwood{at}ucdavis.edu.

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