Protein-Protein Complex Formation Affects the Ni-Fe and Fe-S Centers in the H2-Sensing Regulatory Hydrogenase from Ralstonia eutropha H16

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 RHwt. The RHwt protein transmits its signals to the histidine protein kinase HoxJ, which itself forms a homotetramer and a stable complex with RHwt (RHwt-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 RHwt, protein and the RHwt-HoxJ(wt), complex, we investigated the RHstop protein, which consists of only one HoxB and HoxC unit due to the missing C-terminus of HoxB, as well as RHwt-HoxJ(Delta kinase), 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 RHstop, presumably one Fe-S cluster was lost during the preparation procedure. The coordination of the active site Ni in RHstop, differed from that in RHwt and the RHwt-HoxJ complexes, in which additional Ni-O bonds were detected by XAS. The Ni-O 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.