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J. Biol. Chem., Vol. 279, Issue 45, 46686-46691, November 5, 2004

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The Auxiliary Protein HypX Provides Oxygen Tolerance to the Soluble [NiFe]-Hydrogenase of Ralstonia eutropha H16 by Way of a Cyanide Ligand to Nickel^*

Boris Bleijlevens, Thorsten Buhrke¶, Eddy van der Linden, Bärbel Friedrich¶, and Simon P. J. Albracht||

From the Swammerdam Institute for Life Sciences, Biochemistry, University of Amsterdam, Plantage Muidergracht 12, NL-1018 TV Amsterdam, The Netherlands and ^¶Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Chausseestrasse 117, D-10115 Berlin, Germany

The hypX gene of the facultative lithoautotrophic bacterium Ralstonia eutropha is part of a cassette of accessory genes (the hyp cluster) required for the proper assembly of the active site of the [NiFe]-hydrogenases in the bacterium. A deletion of the hypX gene led to a severe growth retardation under lithoautotrophic conditions with 5 or 15% oxygen, when the growth was dependent on the activity of the soluble NAD⁺-reducing hydrogenase. The enzymatic and infrared spectral properties of the soluble hydrogenase purified from a HypX-negative strain were compared with those from an enzyme purified from a HypX-positive strain. In activity assays under anaerobic conditions both enzyme preparations behaved the same. Under aerobic conditions, however, the mutant enzyme became irreversibly inactivated during H₂ oxidation with NAD⁺ or benzyl viologen as the electron acceptor. Infrared spectra and chemical determination of cyanide showed that one of the four cyanide groups in the wild-type enzyme was missing in the mutant enzyme. The data are consistent with the proposal that the HypX protein is specifically involved in the biosynthetic pathway that delivers the nickel-bound cyanide. The data support the proposal that this cyanide is crucial for the enzyme to function under aerobic conditions.

Received for publication, June 22, 2004 , and in revised form, August 27, 2004.

^* This work was supported by the Netherlands Organization for Scientific Research (NWO), the Deutsche Forschungsgemeinschaft (SFB 498, TP C1), European Union Project BIO4-98-0280, the European Union Cooperation in the field of Scientific and Technical Research (COST), Actions 818 and 841, 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.

These authors contributed equally to this work.

^|| To whom correspondence should be addressed. Tel.: 31-20-5255130; Fax: 31-20-5255124; E-mail: asiem{at}science.uva.nl.

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