title: Maturation of Rhizobium leguminosarum hydrogenase in the presence of oxygen requires the interaction of the chaperone HypC and the scaffolding protein HupK.
abstract: [NiFe] hydrogenases are key enzymes for the energy and redox metabolisms of different microorganisms. Synthesis of these metalloenzymes involves a complex series of biochemical reactions catalyzed by a plethora of accessory proteins, many of them required to synthesize and insert the unique NiFe(CN)2CO cofactor. HypC is an accessory protein conserved in all [NiFe] hydrogenase systems and involved in the synthesis and transfer of the Fe(CN)2CO cofactor precursor. Hydrogenase accessory proteins from bacteria-synthesizing hydrogenase in the presence of oxygen include HupK, a scaffolding protein with a moderate sequence similarity to the hydrogenase large subunit and proposed to participate as an intermediate chaperone in the synthesis of the NiFe cofactor. The endosymbiotic bacterium Rhizobium leguminosarum contains a single hydrogenase system that can be expressed under two different physiological conditions: free-living microaerobic cells (∼ 12 μm O2) and bacteroids from legume nodules (∼ 10-100 nm O2). We have used bioinformatic tools to model HupK structure and interaction of this protein with HypC. Site-directed mutagenesis at positions predicted as critical by the structural analysis have allowed the identification of HupK and HypC residues relevant for the maturation of hydrogenase. Mutant proteins altered in some of these residues show a different phenotype 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.
authors.affiliation: From the Centro de Biotecnología y Genómica de Plantas and Departamento de Biotecnología, Escuela Técnica Superior de Ingenieros Agrónomos, Universidad Politécnica de Madrid, 28223 Pozuelo de Alarcón, Madrid, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Albareda
authors.affiliation: Departamento de Biotecnología, Escuela Técnica Superior de Ingenieros de Montes, Universidad Politécnica de Madrid, 28040 Madrid, Spain, and.
authors.forename: Luis F
authors.initials: LF
authors.lastname: Pacios
authors.affiliation: From the Centro de Biotecnología y Genómica de Plantas and Departamento de Biotecnología, Escuela Técnica Superior de Ingenieros Agrónomos, Universidad Politécnica de Madrid, 28223 Pozuelo de Alarcón, Madrid, Spain.
authors.forename: Hamid
authors.initials: H
authors.lastname: Manyani
authors.affiliation: From the Centro de Biotecnología y Genómica de Plantas and Departamento de Biotecnología, Escuela Técnica Superior de Ingenieros Agrónomos, Universidad Politécnica de Madrid, 28223 Pozuelo de Alarcón, Madrid, Spain.
authors.forename: Luis
authors.initials: L
authors.lastname: Rey
authors.affiliation: From the Centro de Biotecnología y Genómica de Plantas and Departamento de Biotecnología, Escuela Técnica Superior de Ingenieros Agrónomos, Universidad Politécnica de Madrid, 28223 Pozuelo de Alarcón, Madrid, Spain.
authors.forename: Belén
authors.initials: B
authors.lastname: Brito
authors.affiliation: From the Centro de Biotecnología y Genómica de Plantas and Departamento de Biotecnología, Escuela Técnica Superior de Ingenieros Agrónomos, Universidad Politécnica de Madrid, 28223 Pozuelo de Alarcón, Madrid, Spain, Consejo Superior de Investigaciones Científicas, Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, 28223 Pozuelo de Alarcón, Madrid, Spain.
authors.forename: Juan
authors.initials: J
authors.lastname: Imperial
authors.affiliation: From the Centro de Biotecnología y Genómica de Plantas and Departamento de Biotecnología, Escuela Técnica Superior de Ingenieros Agrónomos, Universidad Politécnica de Madrid, 28223 Pozuelo de Alarcón, Madrid, Spain.
authors.forename: Tomás
authors.initials: T
authors.lastname: Ruiz-Argüeso
authors.affiliation: From the Centro de Biotecnología y Genómica de Plantas and Departamento de Biotecnología, Escuela Técnica Superior de Ingenieros Agrónomos, Universidad Politécnica de Madrid, 28223 Pozuelo de Alarcón, Madrid, Spain, jose.palacios@upm.es.
authors.forename: Jose M
authors.initials: JM
authors.lastname: Palacios
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006864:Hydrogenase / Q000378:metabolism*; D008958:Models, Molecular; D010100:Oxygen / Q000378:metabolism*; D011485:Protein Binding; D016961:Rhizobium leguminosarum / Q000201:enzymology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; C108874:HypC protein, Bacteria; C089912:hupK protein, Rhizobium leguminosarum; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D010100:Oxygen
keywords: Hydrogenase; Metalloenzyme; Molecular Modeling; Nitrogen Fixation; Scaffold Protein; [NiFe] Cofactor