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J. Biol. Chem., Vol. 280, Issue 25, 23791-23796, June 24, 2005

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Oxygen Tolerance of the H₂-sensing [NiFe] Hydrogenase from Ralstonia eutropha H16 Is Based on Limited Access of Oxygen to the Active Site^*

Thorsten Buhrke, Oliver Lenz, Norbert Krauss, and Bärbel Friedrich¶

From the Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Chausseestrasse 117, 10115 Berlin, Germany and the Institut für Biochemie, Charité-Universitätsmedizin Berlin, Campus Charité-Mitte, Monbijoustrasse 2, 10117 Berlin, Germany

Hydrogenases, abundant proteins in the microbial world, catalyze cleavage of H₂ into protons and electrons or the evolution of H₂ by proton reduction. Hydrogen metabolism predominantly occurs in anoxic environments mediated by hydrogenases, which are sensitive to inhibition by oxygen. Those microorganisms, which thrive in oxic habitats, contain hydrogenases that operate in the presence of oxygen. We have selected the H₂-sensing regulatory [NiFe] hydrogenase of Ralstonia eutropha H16 to investigate the molecular background of its oxygen tolerance. Evidence is presented that the shape and size of the intramolecular hydrophobic cavities leading to the [NiFe] active site of the regulatory hydrogenase are crucial for oxygen insensitivity. Expansion of the putative gas channel by site-directed mutagenesis yielded mutant derivatives that are sensitive to inhibition by oxygen, presumably because the active site has become accessible for oxygen. The mutant proteins revealed characteristics typical of standard [NiFe] hydrogenases as described for Desulfovibrio gigas and Allochromatium vinosum. The data offer a new strategy how to engineer oxygen-tolerant hydrogenases for biotechnological application.

Received for publication, March 24, 2005 , and in revised form, April 22, 2005.

^* This work was supported by the Deutsche Forschungsgemeinschaft (Sfb498, TP C1) 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.

^¶ To whom correspondence should be addressed. Tel.: 49-30-20938100; Fax: 49-30-20938102; E-mail: baerbel.friedrich{at}rz.hu-berlin.de.

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