The H2 Sensor of Ralstonia eutropha

Abstract

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.

Attenuated total reflectance infrared spectroelectrochemistry at a carbon particle electrode; unmediated redox control of a [NiFe]-hydrogenase solution

“…3C and D are consistent with the presence of Ni-S and Ni-C, respectively. 18 The time course of conversion between these two states after the potential step is displayed in Fig. 3E, indicating that full conversion is achieved in less than 500 s. This implies efficient diffusion of RH within the Nafion-particle network.…”

“…For this study, we select the nickel-iron regulatory hydrogenase (RH) from the 'Knallgas' bacterium Ralstonia eutropha. IR studies have shown the active site of the RH to harbour a standard set of diatomic ligands on the Fe, 17 18,19 The Ni-S state is assigned as Ni II /Fe II and is EPR-silent. 17 Proton-coupled 1-electron reduction leads to the Ni-C state, which is detectable by EPR and is assigned as Ni III /Fe II with a bridging H À (Fig.…”

Probing the Origin of the Metabolic Precursor of the CO Ligand in the Catalytic Center of [NiFe] Hydrogenase

“…A typical IR spectrum of the unlabeled RH stop protein in the oxidized state (27) is shown in Fig. 1A.…”

“…The RH was selected for the present investigations for three reasons: 1) Previous IR analyses uncovered well resolved distinct absorption bands characteristic for CN Ϫ and CO (14,26,27); 2) the RH does not display multiple redox changes as commonly observed with [NiFe] hydrogenases (27); and 3) maturation of the RH requires fewer steps than that of the membranebound counterpart (27). In this study, we have screened a number of 13 C-labeled compounds for their potential to act as direct or indirect precursors of the CO ligand in [NiFe] hydrogenase.…”

Molecular Biology of Microbial Hydrogenases

“…Under these growth conditions, expression of H 2 ase is included in a complex network Vignais et al, 1997Vignais et al, , 2000Vignais et al, , 2002Colbeau et al, 1998;Elsen et al, 2003. Lenz andFriedrich, 1998;Kleihues et al, 2000;Bernhard et al, 2001;Buhrke et al, 2001Buhrke et al, , 2002Lenz et al, 2002;. Elsen et al, 1996.…”