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J. Biol. Chem., Vol. 282, Issue 22, 16177-16186, June 1, 2007

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The Role of Complex Formation between the Escherichia coli Hydrogenase Accessory Factors HypB and SlyD^*

From the Department of Chemistry, University of Toronto, Toronto, Ontario M5S 3H6, Canada

The Escherichia coli protein SlyD is a member of the FK-506-binding protein family of peptidylprolyl isomerases. In addition to its peptidylprolyl isomerase domain, SlyD is composed of a molecular chaperone domain and a C-terminal tail rich in potential metal-binding residues. SlyD interacts with the [NiFe]-hydrogenase accessory protein HypB and contributes to nickel insertion during biosynthesis of the hydrogenase metallocenter. This study examines the HypB-SlyD complex and its significance in hydrogenase activation. Protein variants were prepared to delineate the interface between HypB and SlyD. Complex formation requires the HypB linker region located between the high affinity N-terminal Ni(II) site and the GTPase domain of the protein. In the case of SlyD, the deletion of a short loop in the chaperone domain abrogates the interaction with HypB. Mutations in either protein that disrupt complex formation in vitro also result in deficient hydrogenase production in vivo, indicating that the contact between HypB and SlyD is important for hydrogenase maturation. Surprisingly, SlyD stimulates release of nickel from the high affinity Ni(II)-binding site of HypB, an activity that is also disrupted by mutations that affect complex formation. Furthermore, a SlyD truncation lacking the C-terminal metal-binding tail still interacts with HypB but is deficient in stimulating metal release and is not functional in vivo. These results suggest that SlyD could activate metal release from HypB during metallation of the [NiFe] hydrogenase.

Received for publication, November 22, 2006 , and in revised form, March 13, 2007.

^* This work was supported in part by funding from the Canadian Institutes of Health Research and the Petroleum Research Fund (American Chemical Society). 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 Supported by a postdoctoral fellowship from the Natural Sciences and Engineering Research Council of Canada.

² Supported by a Canada Research Chair. To whom correspondence should be addressed: Dept. of Chemistry, University of Toronto, 80 St. George St., Toronto, Ontario M5S 3H6, Canada. Tel.: 416-978-3568; E-mail: dzamble{at}chem.utoronto.ca.

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