In:
ChemPhysChem, Wiley, Vol. 11, No. 6 ( 2010-04-26), p. 1297-1306
Abstract:
The regulatory Ni–Fe hydrogenase (RH) from the H 2 ‐oxidizing bacterium Ralstonia eutropha functions as an oxygen‐resistant hydrogen sensor, which is composed of the large, active‐site‐containing HoxC subunit and the small subunit HoxB carrying Fe–S clusters. In vivo, the HoxBC subunits form a dimer designated as RH wt . The RH wt protein transmits its signals to the histidine protein kinase HoxJ, which itself forms a homotetramer and a stable complex with RH wt (RH wt –HoxJ wt ), located in the cytoplasm. In this study, we used X‐ray absorption (XAS), electron paramagnetic resonance (EPR), and Fourier transform infrared (FTIR) spectroscopy to investigate the impact of various complexes between RH and HoxJ on the structural and electronic properties of the Ni–Fe active site and the Fe–S clusters. Aside from the RH wt protein and the RH wt –HoxJ wt complex, we investigated the RH stop protein, which consists of only one HoxB and HoxC unit due to the missing C‐terminus of HoxB, as well as RH wt –HoxJ Δkinase , in which the histidine protein kinase lacks the transmitter domain. All constructs reacted with H 2 , leading to the formation of the EPR‐detectable Ni III ‐C state of the active site and to the reduction of Fe–S clusters detectable by XAS, thus corroborating that H 2 cleavage is independent of the presence of the HoxJ protein. In RH stop , presumably one Fe–S cluster was lost during the preparation procedure. The coordination of the active site Ni in RH stop differed from that in RH wt and the RH wt –HoxJ complexes, in which additional NiO bonds were detected by XAS. The NiO bonds caused only very minor changes of the EPR g ‐values of the Ni‐C and Ni‐L states and of the IR vibrational frequencies of the diatomic CN − and CO ligands at the active‐site Fe ion. Both one Fe–S cluster in HoxB and an oxygen‐rich Ni coordination seem to be stabilized by RH dimerization involving the C‐terminus of HoxB and by complex formation with HoxJ.
Type of Medium:
Online Resource
ISSN:
1439-4235
,
1439-7641
DOI:
10.1002/cphc.200901007
Language:
English
Publisher:
Wiley
Publication Date:
2010
detail.hit.zdb_id:
2025223-7
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