The H2-sensing complex of Ralstonia eutropha:: interaction between a regulatory [NiFe] hydrogenase and a histidine protein kinase

Two [NiFe] hydrogenases enable the proteobacterium Ralstonia eutropha H16 to grow on molecular hydrogen as the sole energy source. A third [NiFe] hydrogenase (RH) acts as an H-2 sensor in a multiple component signal transduction chain that controls hydrogenase gene transcription. The RH forms a dimeric heterodimer (HoxBC)(2) in which HoxC contains the H-2-sensing active site and HoxB the electron-transferring components including an organic, not yet identified redox cofactor. This oligomer forms a tight complex with the histidine protein kinase HoxJ. Both the sensor and the kinase were analysed by mutagenesis for functional domains that are instrumental in H-2 signal transmission. A mutant deleted for a C-terminal peptide of 55 amino acids in HoxB lost its H-2-sensing ability but still catalysed H-2 oxidation. The mutant protein failed to form the dimeric heterodimer and a complex with HoxJ. The organic redox cofactor was no longer detectable in the truncated sensor. H-2 sensing was also abolished by deletion of the PAS domain of HoxJ, indicating that this domain is involved in signal transduction. A truncated version of HoxJ consisting of only the input domain of the kinase was still capable of forming a complex with the RH. Mass determination of the purified HoxJ protein revealed that the kinase forms a homotetramer. The unique oligomeric structure of the H-2-sensing complex with respect to its regulatory function is discussed.