Characterization of the hydrogen-deuterium exchange activities of the energy-transducing HupSL hydrogenase and H-2-signaling HupUV hydrogenase in Rhodobacter capsulatus

Rhodobacter capsulatus synthesizes two homologous protein complexes capable of activating molecular H-2, a membrane-bound [NiFe] hydrogenase (HupSL) l inked to the respiratory chain, and an H-2 sensor encoded by the hupUV gene s. The activities of hydrogen-deuterium (H-D) exchange catalyzed by the hup SL-encoded and the hupUV-encoded enzymes in the presence of D-2 and H2O wer e studied comparatively. Whereas HupSL is in the membranes, HupUV activity was localized in the soluble cytoplasmic fraction. Since the hydrogenase ge ne cluster of R. capsulatus contains a gene homologous to hoxH, which encod es the large subunit of NAD-linked tetrameric soluble hydrogenases, the chr omosomal hoxH gene was inactivated and hoxH mutants were used to demonstrat e 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 a nd 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, t he product of the accessory hypD gene was shown to be necessary for the syn thesis of active HupUV enzyme. The kinetics of HD and H-2 formed in exchang e with D-2 by HupUV point to a restricted access of protons and gasses to t he active site. Measurement of concentration changes in D-2, HD, and H-2 by mass spectrometry showed that, besides the H-D exchange reaction, HupUV ox idized H-2 with benzyl viologen, produced H-2 with reduced methyl viologen, and demonstrated true hydrogenase activity. Therefore, not only with respe ct to its H-2 signaling function in the cell, but also to its catalytic pro perties, the HupUV enzyme represents a distinct class of hydrogenases.