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J. Biol. Chem., Vol. 283, Issue 42, 28721-28728, October 17, 2008

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Dual Roles of an Essential Cysteine Residue in Activity of a Redox-regulated Bacterial Transcriptional Activator^*

From the Department of Biological Chemistry, University of Michigan, Ann Arbor, Mighigan 48109-0606

CprK from Desulfitobacterium dehalogenans is the first characterized transcriptional regulator of anaerobic dehalorespiration and is controlled at two levels: redox and effector binding. In the reduced state and in the presence of chlorinated aromatic compounds, CprK positively regulates expression of the cpr gene cluster. One of the products of the cpr gene cluster is CprA, which catalyzes the reductive dehalogenation of chlorinated aromatic compounds. Redox regulation of CprK occurs through a thiol/disulfide redox switch, which includes two classes of cysteine residues. Under oxidizing conditions, Cys¹¹ and Cys²⁰⁰ form an intermolecular disulfide bond, whereas Cys¹⁰⁵ and Cys¹¹¹ form an intramolecular disulfide. Here, we report that Cys¹¹ is involved in redox inactivation in vivo. Upon replacement of Cys¹¹ with serine, alanine, or aspartate, CprK loses its DNA binding activity. C11A is unstable; however, circular dichroism studies demonstrate that the stability and overall secondary structures of CprK and the C11S and C11D variants are similar. Furthermore, effector binding remains intact in the C11S and C11D variants. However, fluorescence spectroscopic results reveal that the tertiary structures of the C11S and C11D variants differ from that of the wild type protein. Thus, Cys¹¹ plays a dual role as a redox switch and in maintaining the correct tertiary structure that promotes DNA binding.

Received for publication, January 24, 2008 , and in revised form, August 6, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant 1P20RR17675. This work was also supported by National Science Foundation Grant GM 39451 (to S. W. R.). 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 Fig. S1-S3.

¹ To whom correspondence should be addressed: Dept. of Biological Chemistry, University of Michigan, University of Michigan Medical School, 5301 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0606. Tel.: 734-615-4621; Fax: 734-763-4581; E-mail: sragsdal{at}umich.edu.

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