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J. Biol. Chem., Vol. 279, Issue 1, 274-281, January 2, 2004

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Characterization of the Menaquinone Reduction Site in the Diheme Cytochrome b Membrane Anchor of Wolinella succinogenes NiFe-hydrogenase^*

Roland Gross, René Pisa, Monica Sänger, C. Roy D. Lancaster, and Jörg Simon¶

From the Institut für Mikrobiologie, Johann Wolfgang Goethe-Universität, Marie-Curie-Str. 9, and the Max-Planck-Institut für Biophysik, Marie-Curie-Str. 15, D-60439 Frankfurt am Main, Germany

The majority of bacterial membrane-bound NiFe-hydrogenases and formate dehydrogenases have homologous membrane-integral cytochrome b subunits. The prototypic NiFe-hydrogenase of Wolinella succinogenes (HydABC complex) catalyzes H₂ oxidation by menaquinone during anaerobic respiration and contains a membrane-integral cytochrome b subunit (HydC) that carries the menaquinone reduction site. Using the crystal structure of the homologous FdnI subunit of Escherichia coli formate dehydrogenase-N as a model, the HydC protein was modified to examine residues thought to be involved in menaquinone binding. Variant HydABC complexes were produced in W. succinogenes, and several conserved HydC residues were identified that are essential for growth with H₂ as electron donor and for quinone reduction by H₂. Modification of HydC with a C-terminal Strep-tag II enabled one-step purification of the HydABC complex by Strep-Tactin affinity chromatography. The tagged HydC, separated from HydAB by isoelectric focusing, was shown to contain 1.9 mol of heme b/mol of HydC demonstrating that HydC ligates both heme b groups. The four histidine residues predicted as axial heme b ligands were individually replaced by alanine in Strep-tagged HydC. Replacement of either histidine ligand of the heme b group proximal to HydAB led to HydABC preparations that contained only one heme b group. This remaining heme b could be completely reduced by quinone supporting the view that the menaquinone reduction site is located near the distal heme b group. The results indicate that both heme b groups are involved in electron transport and that the architecture of the menaquinone reduction site near the cytoplasmic side of the membrane is similar to that proposed for E. coli FdnI.

Received for publication, September 25, 2003 , and in revised form, October 22, 2003.

^* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 472 and the Fonds der Chemischen Industrie. 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.

^¶ To whom correspondence should be addressed. Tel.: 49-69-79829320; Fax: 49-69-79829527; E-mail: j.simon{at}em.uni-frankfurt.de.

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