The role of the twin-arginine motif in the signal peptide encoded by the hydA gene of the hydrogenase from Wolinella succinogenes

The hydABC operon of Wolinella succinogenes encodes the three subunits of t he membrane-integrated Ni-hydrogenase. The catalytic subunit, HydB, is on t he periplasmic side of the membrane. Residues R41 and R42 of the twin-argin ine motif within the signal peptide of the precursor of the iron-sulfur sub unit, HydA, were replaced by two glutamine residues. The corresponding muta nt did not grow with H-2 as the electron donor of anaerobic respiration. Ma ture HydB and the precursor protein of HydA were located exclusively in the cytoplasmic cell fraction of the mutant, which catalyzed the reduction of benzyl viologen by H-2 Suggesting that HydB contained Ni. The HydC protein was located in the membrane fraction of the are homologous to the subunits of the periplasmic hydromutant in wild-type amounts. HydC was purified and was Introduction are homologous to the subunits of the periplasmic hydrosho wn to contain heme. The results suggest that HydA and HydB are translocated across the membrane by the Tat (twin-arginine translocation) system. The t ranslocation of HydA and HydB as well as the maturation of the precursor pr otein of HydA appear to depend on the presence of the twin-arginine motif. In contrast, maturation of HydB, the insertion of HydC into the membrane, a nd heme attachment to HydC are apparently independent of the twin-arginine motif and do not require translocation of the two other hydrogenase subunit s.