Gene Products of the hupGHIJ Operon Are Involved in Maturation of the Iron-Sulfur Subunit of the [NiFe] Hydrogenase from Rhizobium leguminosarum bv. viciae

Abstract

In the present study, we investigate the functions of the hupGHIJ operon in the synthesis of an active [NiFe] hydrogenase in the legume endosymbiont Rhizobium leguminosarum bv. viciae. These genes are clustered with 14 other genes including the hydrogenase structural genes hupSL. A set of isogenic mutants with in-frame deletions (⌬hupG, ⌬hupH, ⌬hupI, and ⌬hupJ) was generated and tested for hydrogenase activity in cultures grown at different oxygen concentrations (0.2 to 2.0%) and in symbiosis with peas. In free-living cultures, deletions in these genes severely reduced hydrogenase activity. The ⌬hupH mutant was totally devoid of hydrogenase activity at any of the O 2 concentration tested, whereas the requirement of hupGIJ for hydrogenase activity varied with the O 2 concentration, being more crucial at higher pO 2 . Pea bacteroids from the mutant strains affected in hupH, hupI, and hupJ exhibited reduced (20 to 50%) rates of hydrogenase activity compared to the wild type, whereas rates were not affected in the ⌬hupG mutant. Immunoblot experiments with HupLand HupS-specific antisera showed that free-living cultures from ⌬hupH, ⌬hupI, and ⌬hupJ mutants synthesized a fully processed mature HupL protein and accumulated an unprocessed form of HupS (pre-HupS). Both the mature HupL and the pre-HupS forms were located in the cytoplasmic fraction of cultures from the ⌬hupH mutant. Affinity chromatography experiments revealed that cytoplasmic pre-HupS binds to the HupH protein before the pre-HupS-HupL complex is formed. From these results we propose that hupGHIJ gene products are involved in the maturation of the HupS hydrogenase subunit.The protein core of [NiFe] hydrogenases is composed of two different subunits. The large subunit contains the catalytic site consisting of a heterobinuclear NiFe metallocenter, and the small subunit from most [NiFe] hydrogenases holds three Fe-S clusters (43). Multiple genes are required for the synthesis of [NiFe] hydrogenases, and most of them are conserved among different bacteria (40,41). The role of proteins encoded by these genes in the synthesis process is only partially known. Analysis of Escherichia coli hydrogenase 3 revealed that Hyp proteins are involved in the biosynthesis of the Ni-Fe cofactor, a process which ends with the processing of the large subunit by an endopeptidase which removes a C-terminal tail from the protein (3). In contrast, no auxiliary proteins have been identified that are required for synthesis of a functional small subunit of the [NiFe] hydrogenases, although most of them contain three iron-sulfur clusters (two 4Fe-4S and one 3Fe-4S) that conduct electrons from the H 2 -activating center in the large subunit to the physiological electron acceptor on the surface of the enzyme (11). Different proteins are known that facilitate the assembly of Fe-S clusters into other Fe-S proteins (29, 44).In symbiosis with peas, Rhizobium leguminosarum bv. viciae strain UPM791 induces an H 2 uptake [NiFe] hydrogenase whose genetic determinants are grouped in a cluster ...

Biosynthesis of Salmonella enterica [NiFe]-hydrogenase-5: probing the roles of system-specific accessory proteins

“…leguminosarum HupH [20] we would expect HydG to interact with the small subunit of Hyd-5, performing an important role in FeS cluster insertion into HydA. This is consistent with our results, since we show that HydG is essential for the production of active Hyd-5 under both low and high O2 conditions.…”

“…Our results are therefore surprising, 20 since we show that HydF is redundant in the biosynthesis of S. enterica Hyd-5, regardless of O2 level. We prove that the resultant enzyme is fully active, and that maturation of the proximal [4Fe3S] cluster, which is unique to O2-tolerant [NiFe]-MBH, has not been negatively impacted by deletion of hydF.…”

“…leguminosarum, HupG [20], it has been determined that the function of these proteins is to assist in the maturation of the FeS-containing protein subunit of the final hydrogenase enzyme complex. The specific role is thought to involve binding of the HyaE-homologue to the Tat signal peptide of the hydrogenase small subunit that is synthesized from the same operon [13,15], perhaps explaining the presence of highly conserved negatively charged residues ( Figure S3) [41].…”

“…A Rh. leguminosarum ΔhupG study showed that the higher the oxygen concentration the more important the function of HupG in hydrogenase maturation [20]. Thus, it is possible to rationalise why the deletion of hyaE had an insignificant effect on the assembly of the anaerobically synthesized enzyme E. coli Hyd-1…”

“…R. eutropha couples H2 oxidation to O2 reduction and must therefore be grown under at least microaerobic conditions, while E. coli only synthesizes MBH under anaerobic conditions. In light of this, insightful studies into hydrogenase maturation have been performed using Rhizobium leguminosarum, a N2-fixing soil bacterium that can be grown under very low and high O2 conditions and produces one [NiFe] MBH [19][20][21]. To complement this previous work, we have used S. enterica as a model system to study hydrogenase assembly.…”