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J. Biol. Chem., Vol. 281, Issue 40, 30186-30194, October 6, 2006

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Function and Structure of the Molybdenum Cofactor Carrier Protein from Chlamydomonas reinhardtii^*

Katrin Fischer¹, Angel Llamas^¶1, Manuel Tejada-Jimenez^¶1, Nils Schrader^||, Jochen Kuper^**, Farid S. Ataya^¶, Aurora Galvan^¶, Ralf R. Mendel, Emilio Fernandez^¶, and Guenter Schwarz²

From the Institute of Plant Biology, Technical University Braunschweig, 38106 Braunschweig, Germany, the ^¶Departamento de Bioquímica y Biología Molecular, Universidad de Córdoba, Córdoba 14071, Spain, the ^||Department of Structural Biology, Max-Planck-Institute of Molecular Physiology, 44247 Dortmund, Germany, the ^**European Molecular Biology Laboratory (EMBL) outstation, 22603 Hamburg, Germany, and the Institute of Biochemistry, University of Cologne, 50674 Cologne, Germany

The molybdenum cofactor (Moco) forms the catalytic site in all eukaryotic molybdenum enzymes and is synthesized by a multistep biosynthetic pathway. The mechanism of transfer, storage, and insertion of Moco into the appropriate apo-enzyme is poorly understood. In Chlamydomonas reinhardtii, a Moco carrier protein (MCP) has been identified and characterized recently. Here we show biochemical evidence that MCP binds Moco as well as the tungstate-substituted form of the cofactor (Wco) with high affinity, whereas molybdopterin, the ultimate cofactor precursor, is not bound. This binding selectivity points to a specific metal-mediated interaction with MCP, which protects Moco and Wco from oxidation with t of 24 and 96 h, respectively. UV-visible spectroscopy showed defined absorption bands at 393, 470, and 570 nm pointing to ene-diothiolate and protein side-chain charge transfer bonds with molybdenum. We have determined the crystal structure of MCP at 1.6Å resolution using seleno-methionated and native protein. The monomer constitutes a Rossmann fold with two homodimers forming a symmetrical tetramer in solution. Based on conserved surface residues, charge distribution, shape, in silico docking studies, structural comparisons, and identification of an anionbinding site, a prominent surface depression was proposed as a Moco-binding site, which was confirmed by structure-guided mutagenesis coupled to substrate binding studies.

Received for publication, April 24, 2006 , and in revised form, June 23, 2006.

The atomic coordinates and structure factors (code 2IZ5, 2IZ6, and 2IZ7) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by the Deutsche Forschungsgemeinschaft (to R. R. M. and G. S.), the European Union grant (to R. R. M.), the Ministerio de Educación y Ciencia (to E. F.), Junta de Andalucía (to A. G.), and the Fonds der Chemischen Industrie (to G. S.). 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 These authors equally contributed to the work and are listed in alphabetical order.

² To whom correspondence should be addressed: Guenter Schwarz, Institut für Biochemie, Universität zu Köln, Otto-Fischer-Str. 12-14, 50674 Köln, Germany. Tel.: 49-221-470-6432; Fax: 49-221-470-6731; E-mail: gschwarz{at}uni-koeln.de.

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