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.
Institut für Biologie, Humboldt-Universität zu Berlin, Chausseestrasse 117, 10115 Berlin, Germany.
M
M
Bernhard
T
T
Buhrke
B
B
Bleijlevens
A L
AL
De Lacey
V M
VM
Fernandez
S P
SP
Albracht
B
B
Friedrich
D002852:Chromatography, Ion Exchange; D020118:Cupriavidus necator / Q000254:growth & development / Q000378:metabolism*; D003593:Cytoplasm / Q000201:enzymology; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D000071677:Histidine Kinase; D006859:Hydrogen / Q000378:metabolism*; D006864:Hydrogenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D010084:Oxidation-Reduction; D011494:Protein Kinases / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared
D006859:Hydrogen; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D011494:Protein Kinases; D000071677:Histidine Kinase