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J. Biol. Chem., Vol. 281, Issue 36, 26382-26390, September 8, 2006

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Transcriptional Activation of Dehalorespiration

IDENTIFICATION OF REDOX-ACTIVE CYSTEINES REGULATING DIMERIZATION AND DNA BINDING^*

From the Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588-0664

Desulfitobacterium dehalogenans can use chlorinated aromatics including polychlorinated biphenyls as electron acceptors in a process called dehalorespiration. Expression of the cpr gene cluster involved in this process is regulated by CprK, which is a member of the CRP/FNR (cAMP-binding protein/fumarate nitrate reduction regulatory protein) family of helix-turn-helix transcriptional regulators. High affinity interaction of the chlorinated aromatic compound with the effector domain of CprK triggers binding of CprK to an upstream target DNA sequence, which leads to transcriptional activation of the cpr gene cluster. When incubated with oxygen or diamide, CprK undergoes inactivation; subsequent treatment with dithiothreitol restores activity. Using mass spectrometry, this study identifies two classes of redox-active thiol groups that form disulfide bonds upon oxidation. Under oxidative conditions, Cys¹⁰⁵, which is conserved in FNR and most other CprK homologs, forms an intramolecular disulfide bond with Cys¹¹¹, whereas an intermolecular disulfide bond is formed between Cys¹¹ and Cys²⁰⁰. SDS-PAGE and site-directed mutagenesis experiments indicate that the Cys¹¹/Cys²⁰⁰ disulfide bond links two CprK subunits in an inactive dimer. Isothermal calorimetry and intrinsic fluorescence quenching studies show that oxidation does not change the affinity of CprK for the effector. Therefore, reversible redox inactivation is manifested at the level of DNA binding. Our studies reveal a strategy for limiting expression of a redox-sensitive pathway by using a thiol-based redox switch in the transcription factor.

Received for publication, March 7, 2006 , and in revised form, July 10, 2006.

^* This work was supported by a grant from National Science Foundation Grant MCB0211730 and in part by National Institutes of Health Grant 1P20RR17675. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ These authors contributed equally to this work.

² Present address: Huntsman Cancer Institute, Dept. of Oncological Sciences, Rm. 4210, 4L, 2000 Circle of Hope, University of Utah, Salt Lake City, UT 84112-5550.

³ To whom correspondence should be addressed: Dept. of Biochemistry, Beadle Center, 19th & Vine St., University of Nebraska, Lincoln, NE 68588-0664. Tel.: 402-472-2943; Fax: 402-472-4961; E-mail: sragsdale1{at}unl.edu.

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