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J. Biol. Chem., Vol. 280, Issue 20, 19488-19495, May 20, 2005

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Reduction of Unusual Iron-Sulfur Clusters in the H₂-sensing Regulatory Ni-Fe Hydrogenase from Ralstonia eutropha H16^*

Thorsten Buhrke, Simone Löscher¶, Oliver Lenz, Eberhard Schlodder||, Ingo Zebger||, Lars K. Andersen||, Peter Hildebrandt||, Wolfram Meyer-Klaucke**, Holger Dau¶, Bärbel Friedrich, and Michael Haumann¶

From the Humboldt-Universität zu Berlin, Institut für Biologie/Mikrobiologie, Chausseestrasse 117, D-10115 Berlin, Germany, ^¶Freie Universität Berlin, FB Physik, Arnimallee 14, D-14195 Berlin, Germany, ^||Technische Universität Berlin, Institut für Chemie, Max-Volmer-Laboratorium für Biophysikalische Chemie, Sekretariat. PC14, Strasse des 17, Juni 135, D-10623 Berlin, Germany, and ^**DESY, EMBL Outstation, Notkestrasse 85, D-22603 Hamburg, Germany

The regulatory Ni-Fe hydrogenase (RH) from Ralstonia eutropha functions as a hydrogen sensor. The RH consists of the large subunit HoxC housing the Ni-Fe active site and the small subunit HoxB containing Fe-S clusters. The heterolytic cleavage of H₂ at the Ni-Fe active site leads to the EPR-detectable Ni-C state of the protein. For the first time, the simultaneous but EPR-invisible reduction of Fe-S clusters during Ni-C state formation was demonstrated by changes in the UV-visible absorption spectrum as well as by shifts of the iron K-edge from x-ray absorption spectroscopy in the wild-type double dimeric RH_WT [HoxBC]₂ and in a monodimeric derivative designated RH_stop lacking the C-terminal 55 amino acids of HoxB. According to the analysis of iron EXAFS spectra, the Fe-S clusters of HoxB pronouncedly differ from the three Fe-S clusters in the small subunits of crystallized standard Ni-Fe hydrogenases. Each HoxBC unit of RH_WT seems to harbor two [2Fe-2S] clusters in addition to a 4Fe species, which may be a [4Fe-3S-3O] cluster. The additional 4Fe-cluster was absent in RH_stop. Reduction of Fe-S clusters in the hydrogen sensor RH may be a first step in the signal transduction chain, which involves complex formation between [HoxBC]₂ and tetrameric HoxJ protein, leading to the expression of the energy converting Ni-Fe hydrogenases in R. eutropha.

Received for publication, January 18, 2005 , and in revised form, February 23, 2005.

^* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 498 (Projects C1, C6, C8, and A8) and by 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.

These authors contributed equally to this work.

To whom correspondence should be addressed. Tel.: 49-30-8385-6101; Fax: 49-30-8385-6299; E-mail: haumann{at}physik.fu-berlin.de.

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