M. Bernhard et al., The H-2 sensor of Ralstonia eutropha - Biochemical characteristics, spectroscopic properties, and its interaction with a histidine protein kinase, J BIOL CHEM, 276(19), 2001, pp. 15592-15597
Previous genetic studies have revealed a multicomponent signal transduction
chain, consisting of an H-2 sensor, a histidine protein kinase, and a resp
onse regulator, which controls hydrogenase gene transcription in the proteo
bacterium 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 hy
drogenases and insensitive to oxygen, carbon monoxide, and acetylene, Inter
estingly, 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 sim
ilar to that of standard [NiFe]-hydrogenases. It is suggested that the prot
ein 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 e
ven 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 possi
ble involvement of this chromophore in signal transduction is discussed.