Rhodobacter capsulatus synthesizes two homologous protein complexes capable of activating molecular H₂, a membrane-bound [NiFe] hydrogenase (HupSL) linked to the respiratory chain, and an H₂ sensor encoded by the hupUV genes. The activities of hydrogen-deuterium (H-D) exchange catalyzed by the hupSL-encoded and the hupUV-encoded enzymes in the presence of D₂ and H₂O were studied comparatively. Whereas HupSL is in the membranes, HupUV activity was localized in the soluble cytoplasmic fraction. Since the hydrogenase gene cluster of R. capsulatus contains a gene homologous to hoxH, which encodes the large subunit of NAD-linked tetrameric soluble hydrogenases, the chromosomal hoxH gene was inactivated and hoxH mutants were used to demonstrate the H-D exchange activity of the cytoplasmic HupUV protein complex. The H-D exchange reaction catalyzed by HupSL hydrogenase was maximal at pH 4.5 and inhibited by acetylene and oxygen, whereas the H-D exchange catalyzed by the HupUV protein complex was insensitive to acetylene and oxygen and did not vary significantly between pH 4 and pH 11. Based on these properties, the product of the accessory hypD gene was shown to be necessary for the synthesis of active HupUV enzyme. The kinetics of HD and H₂ formed in exchange with D₂ by HupUV point to a restricted access of protons and gasses to the active site. Measurement of concentration changes in D₂, HD, and H₂ by mass spectrometry showed that, besides the H-D exchange reaction, HupUV oxidized H₂ with benzyl viologen, produced H₂ with reduced methyl viologen, and demonstrated true hydrogenase activity. Therefore, not only with respect to its H₂ signaling function in the cell, but also to its catalytic properties, the HupUV enzyme represents a distinct class of hydrogenases.