[NiFe] hydrogenases are key enzymes for the energy and redox metabolisms of different microorganisms. depending on the physiological condition tested. Modeling of HypC also predicts the existence of a stable HypC dimer whose presence was also demonstrated by immunoblot analysis. This study widens our understanding on the mechanisms for metalloenzyme biosynthesis in the presence of oxygen. hydrogenase 3 has shown that cyano ligands are synthesized from carbamoyl phosphate (9) through the concerted action of HypF and HypE proteins (10) and transmitted to an iron atom exposed on a HypCD complex (11, 12). The N-terminal cysteine residue of HypC (Cys-2) Desacetyl asperulosidic acid supplier is essential for the interaction with HypD and HycE (11, 12). Infrared spectroscopy analysis has shown that HypCD complexes from and exhibit bands characteristic of diatomic CO and CN ligands (12, 13). More recent studies have demonstrated that HypD acts as a scaffolding protein in which the precursor cofactor is formed (14). A recent report (15) suggests that HypC is able to bind iron and CO2 and probably delivers both to HypD scaffold protein, where reduction of CO2 occurs. Two HypC residues (Cys-2 and His-51 in the protein) are essential for binding CO2 and Desacetyl asperulosidic acid supplier iron and, according to molecular dynamics calculations, these residues might Desacetyl asperulosidic acid supplier also participate in binding the Fe(CN)2CO cofactor precursor (16). In the model system, HypC transfers this cofactor precursor to HycE, the large structural subunit in this system (17). Once all ligands are in place, proteins HypA, HypB, and SlyD mediate nickel incorporation into the active site (18). HypC chaperone dissociates from HycE after nickel insertion, and the large subunit is proteolytically processed by a highly specific, nickel-dependent protease (19). Some diazotrophic bacteria induce a [NiFe] hydrogenase that catalyzes the oxidation of hydrogen produced by nitrogenase during the nitrogen fixation process. The recycling of H2 is especially relevant in legume symbiosis Rabbit Polyclonal to SREBP-1 (phospho-Ser439) because it has the potential to increase the energy efficiency of nitrogen fixation, and increments in plant productivity associated to this trait have been demonstrated (20, 21). In bv. the genetic determinants involved in the biosynthesis of the hydrogenase are clustered in the symbiotic plasmid and include 18 genes Desacetyl asperulosidic acid supplier (promoter was replaced by the Fnr-dependent promoter, allowing the expression of hydrogenase in microaerobic vegetative cells (24). The hydrogenase system includes two proteins, HupF and HupK, not present in the model described above. These two proteins are conserved in other hydrogenase systems in which the enzyme is synthesized in the presence of O2. On the basis of its structural homology to hydrogenase large subunit, HupK was proposed as a scaffolding protein (25), and studies in have demonstrated that HoxV, a HupK homolog, is able to bind the cofactor precursor as an intermediate step to its insertion into the large structural subunit HoxG (26). HoxL, the HupF homolog in HupF acts as a chaperone to stabilize the large subunit HupL when hydrogenase is synthesized in the presence of O2 (27). The fact that contains a single [NiFe] hydrogenase gene cluster makes this system particularly well suited for studying the molecular basis of biosynthesis of the enzyme as compared with other systems having multiple hydrogenases. Furthermore, the availability of two expression conditions with different oxygen tensions (1% O2 in microaerobic cultures and virtually anaerobic within legume nodules) allows the identification of oxygen-dependent functions during the biosynthetic process (28). In this work we demonstrate that the accessory protein HupK has a relevant role in the biosynthesis of hydrogenase in strains were routinely grown at 28 C in yeast mannitol broth (YMB),4 Tryptone-yeast extract, or minimal media (24). DH5 was used for standard cloning procedures, and S17.1 was used as the donor for conjugative plasmid transfer between and cells were carried out by standard methods (31). Oligonucleotides used as primers are listed in Table 2. TABLE 2 Primers used in this work To generate the HypC::from the pALPF1 plasmid using primers TAGYC1-TAGYC2. The resulting plasmid (pALPF36) harbors a hydrogenase gene cluster encoding a gene in microaerobically grown cultures of in a way compatible with Hup expression from pALPF1 derivatives, a pBBR1MCS derivative plasmid (pPM502).