The H2 sensor of Ralstonia eutropha -: Biochemical characteristics, spectroscopic properties, and its interaction with a histidine protein kinase

Previous genetic studies have revealed a multicomponent signal transduction chain, consisting of an H-2 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 H-2 sensor and demonstrated that the purified protein forms a complex with the histidine protein kinase, Biochemical and spectroscopic analysis revealed that the H-2 sensor is a cytoplasmic [NiFe]-hydrogenase with unique features. The H-2-oxidizing activity was 2 orders of magnitude lower than that of standard hydrogenases and insensitive to oxygen, carbon monoxide, and acetylene, Interestingly, only H-2 production but no HD formation was detected in the D-2/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 H-2 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 H-2. The possible involvement of this chromophore in signal transduction is discussed.