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J. Biol. Chem., Vol. 276, Issue 19, 15592-15597, May 11, 2001

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The H₂ Sensor of Ralstonia eutropha
BIOCHEMICAL CHARACTERISTICS, SPECTROSCOPIC PROPERTIES, AND ITS INTERACTION WITH A HISTIDINE PROTEIN KINASE^*

Michael Bernhard, Thorsten Buhrke, Boris Bleijlevens^§, Antonio L. De Lacey^¶, Victor M. Fernandez^¶, Simon P. J. Albracht^§, and Bärbel Friedrich

From the  Institut für Biologie, Humboldt-Universität zu Berlin, Chausseestrasse 117, 10115 Berlin, Germany, the ^§ Swammerdam Institute for Life Sciences, Department of Biochemistry, University of Amsterdam, Plantage Muidergracht 12, NL-1018 TV Amsterdam, The Netherlands, and the ^¶ Instituto de Catalisis, CSIC, Campus Universidad Autonoma-Cantoblanco, 28049 Madrid, Spain

Previous genetic studies have revealed a multicomponent signal transduction chain, consisting of an H₂ sensor, a histidine protein kinase, and a response regulator, which controls hydrogenase gene transcription in the proteobacterium Ralstonia eutropha. In this study, we isolated the H₂ sensor and demonstrated that the purified protein forms a complex with the histidine protein kinase. Biochemical and spectroscopic analysis revealed that the H₂ sensor is a cytoplasmic [NiFe]-hydrogenase with unique features. The H₂-oxidizing activity was 2 orders of magnitude lower than that of standard hydrogenases and insensitive to oxygen, carbon monoxide, and acetylene. Interestingly, only H₂ production but no HD formation was detected in the D₂/H⁺ exchange assay. Fourier transform infrared data showed an active site similar to that of standard [NiFe]-hydrogenases. It is suggested that the protein environment accounts for a restricted gas diffusion and for the typical kinetic parameters of the H₂ sensor. EPR analysis demonstrated that the [4Fe-4S] clusters within the small subunit were not reduced under hydrogen even in the presence of dithionite. Optical spectra revealed the presence of a novel, redox-active, n = 2 chromophore that is reduced by H₂. The possible involvement of this chromophore in signal transduction is discussed.

This paper is dedicated to Prof. Ernst-G. Jäger on the occasion of his 65th birthday on May 5, 2001.

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