Impact of alterations near the [NiFe] active site on the function of the H2 sensor from Ralstonia eutropha

In proteobacteria capable of H2 oxidation under (micro)aerobic conditions, hydrogenase gene expression is often controlled in response to the availability of H2. The H2-sensing signal transduction pathway consists of a heterodimeric regulatory [NiFe]-hydrogenase (RH), a histidine protein kinase and a response regulator. To gain insights into the signal transmission from the Ni–Fe active site in the RH to the histidine protein kinase, conserved amino acid residues in the L0 motif near the active site of the RH large subunit of Ralstonia eutropha H16 were exchanged. Replacement of the strictly conserved Glu13 (E13N, E13L) resulted in loss of the regulatory, H2-oxidizing and D2/H+ exchange activities of the RH. According to EPR and FTIR analysis, these RH derivatives contained fully assembled [NiFe] active sites, and para-/ortho-H2 conversion activity showed that these centres were still able to bind H2. This indicates that H2 binding at the active site is not sufficient for the regulatory function of H2 sensors. Replacement of His15, a residue unique in RHs, by Asp restored the consensus of energy-linked [NiFe]-hydrogenases. The respective RH mutant protein showed only traces of H2-oxidizing activity, whereas its D2/H+-exchange activity and H2-sensing function were almost unaffected. H2-dependent signal transduction in this mutant was less sensitive to oxygen than in the wild-type strain. These results suggest that H2 turnover is not crucial for H2 sensing. It may even be detrimental for the function of the H2 sensor under high O2 concentrations.