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J. Biol. Chem., Vol. 283, Issue 27, 18861-18872, July 4, 2008

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X-ray Structure of the [FeFe]-Hydrogenase Maturase HydE from Thermotoga maritima^*

Yvain Nicolet, Jon K. Rubach¹, Matthew C. Posewitz^¶, Patricia Amara, Carole Mathevon, Mohamed Atta, Marc Fontecave, and Juan C. Fontecilla-Camps²

From the Laboratoire de Cristallographie et Cristallogenèse des Protéines, Institut de Biologie Structurale J.P. Ebel, Commissariat à l'Energie Atomique, CNRS, Université J. Fourier, 41 Rue J. Horowitz, 38027 Grenoble Cedex 1, France, Laboratoire de Chimie et Biologie des Métaux, Institut de Recherches en Technologies et Sciences pour le Vivant, Commissariat à l'Energie Atomique, CNRS, UMR 5249, Université J. Fourier, 17 Avenue des Martyrs, 38054 Grenoble Cedex 09, France, and ^¶Colorado School of Mines, Department of Chemistry and Geochemistry, Golden, Colorado 80401

Maturation of the [FeFe]-hydrogenase active site depends on at least the expression of three gene products called HydE, HydF, and HydG. We have solved the high resolution structure of recombinant, reconstituted S-adenosine-L-methionine-dependent HydE from Thermotoga maritima. Besides the conserved [Fe₄S₄] cluster involved in the radical-based reaction, this HydE was reported to have a second [Fe₄S₄] cluster coordinated by three Cys residues. However, in our crystals, depending on the reconstitution and soaking conditions, this second cluster is either a [Fe₂S₂] center, with water occupying the fourth ligand site or is absent. We have carried out site-directed mutagenesis studies on the related HydE from Clostridium acetobutylicum, along with in silico docking and crystal soaking experiments, to define the active site region and three anion-binding sites inside a large, positive cavity, one of which binds SCN^- with high affinity. Although the overall triose-phosphate isomerase-barrel structure of HydE is very similar to that of biotin synthase, the residues that line the internal cavity are significantly different in the two enzymes.

Received for publication, February 12, 2008 , and in revised form, March 27, 2008.

The atomic coordinates and structure factors (codes 3CIW and 3CIX) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* 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 Table 1 and Figs. 1–4.

¹ Present address: Life Sciences Institute, University of Michigan, 210 Washtenaw Ave., Ann Arbor, MI 48109.

² To whom correspondence should be addressed. Tel.: 33-4-38785920; Fax: 33-4-38785122; E-mail: juan.fontecilla{at}ibs.fr.

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