Two energy-generating hydrogenases enable the aerobic hydrogen bacterium Ralstonia eutropha (formerly Alcaligenes eutrophus) to use molecular hydrogen as the sole energy source. The complex synthesis of the nickel-iron-containing enzymes has to be efficiently regulated in response to H 2 , which is available in low amounts in aerobic environments. H 2 sensing in R. eutropha is achieved by a hydrogenase-like protein which controls the hydrogenase gene expression in concert with a two-component regulatory system. In this study we show that the H 2 sensor of R. eutropha is a cytoplasmic protein. oxidation and electron transfer processes are necessary for signaling. The regulatory role of the H 2 -sensing hydrogenase of R. eutropha, which has also been investigated in other hydrogen-oxidizing bacteria, is intimately correlated with a set of typical structural features. Thus, the family of H 2 sensors represents a novel subclass of [NiFe] hydrogenases denoted as the "regulatory hydrogenases."Molecular hydrogen is frequently used as an energy source by diverse prokaryotic organisms. Many of these bacterial and archaeal species harbor multiple hydrogenases which mediate heterolytic cleavage of H 2 into 2 H ϩ and 2 e Ϫ .[NiFe] hydrogenases are the most dominant enzymes, representing a fairly conserved family of proteins, composed of at least a large active site-containing subunit and a small electron-transferring subunit which bears one to three FeS clusters (1, 2, 18).The facultative chemolithoautotrophic proteobacterium Ralstonia eutropha H16 (formerly Alcaligenes eutrophus [7]) harbors two energy-generating [NiFe] hydrogenases, a membrane-bound enzyme (MBH) and a cytoplasmic enzyme (SH). The MBH is primarily involved in electron transport-coupled phosphorylation, whereas the SH is able to reduce NAD and thus provides the cell with reducing equivalents (38, 40). The composition of the MBH resembles the prototype of [NiFe] hydrogenases whose atomic structure has been resolved by X-ray analysis (50). The two subunits of the R. eutropha MBH, encoded by hoxK and hoxG, are anchored to the outer face of the cytoplasmic membrane via a b-type cytochrome (4). The SH, encoded by hoxF, hoxU, hoxY, and hoxH, contains an FeS-flavoprotein in addition to the hydrogenase moiety (30). Mutants disrupted in either one of the two hydrogenases maintain their ability to grow on H 2 , which indicates that the two enzymes can replace each other physiologically (23).The hydrogenase-related genes of R. eutropha are organized in the MBH and the SH operons, which are regulated coordinately (42). The MBH operon comprises 10 MBH-specific genes in addition to a set of accessory genes whose products are involved in the complex posttranslational maturation of the hydrogenases and the regulation of both the MBH and the SH operon (5,11,24,41). The SH operon harbors the structural genes of the NAD-reducing hydrogenase together with a set of accessory genes which code for maturation proteins (45,47,52).Hydrogenase gene expression is controlled by the m...