title: Intact functional fourteen-subunit respiratory membrane-bound [NiFe]-hydrogenase complex of the hyperthermophilic archaeon Pyrococcus furiosus.
abstract: The archaeon Pyrococcus furiosus grows optimally at 100 °C by converting carbohydrates to acetate, CO2, and H2, obtaining energy from a respiratory membrane-bound hydrogenase (MBH). This conserves energy by coupling H2 production to oxidation of reduced ferredoxin with generation of a sodium ion gradient. MBH is encoded by a 14-gene operon with both hydrogenase and Na(+)/H(+) antiporter modules. Herein a His-tagged MBH was expressed in P. furiosus and the detergent-solubilized complex purified under anaerobic conditions by affinity chromatography. Purified MBH contains all 14 subunits by electrophoretic analysis (13 subunits were also identified by mass spectrometry) and had a measured iron:nickel ratio of 15:1, resembling the predicted value of 13:1. The as-purified enzyme exhibited a rhombic EPR signal characteristic of the ready nickel-boron state. The purified and membrane-bound forms of MBH both preferentially evolved H2 with the physiological donor (reduced ferredoxin) as well as with standard dyes. The O2 sensitivities of the two forms were similar (half-lives of ∼ 15 h in air), but the purified enzyme was more thermolabile (half-lives at 90 °C of 1 and 25 h, respectively). Structural analysis of purified MBH by small angle x-ray scattering indicated a Z-shaped structure with a mass of 310 kDa, resembling the predicted value (298 kDa). The angle x-ray scattering analyses reinforce and extend the conserved sequence relationships of group 4 enzymes and complex I (NADH quinone oxidoreductase). This is the first report on the properties of a solubilized form of an intact respiratory MBH complex that is proposed to evolve H2 and pump Na(+) ions.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602-7229.
authors.forename: Patrick M
authors.initials: PM
authors.lastname: McTernan
authors.affiliation: From the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602-7229.
authors.forename: Sanjeev K
authors.initials: SK
authors.lastname: Chandrayan
authors.affiliation: From the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602-7229.
authors.forename: Chang-Hao
authors.initials: CH
authors.lastname: Wu
authors.affiliation: From the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602-7229.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Vaccaro
authors.affiliation: From the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602-7229.
authors.forename: W Andrew
authors.initials: WA
authors.lastname: Lancaster
authors.affiliation: the Department of Physiology, John Hopkins University School of Medicine, Baltimore, Maryland 21205, and.
authors.forename: Qingyuan
authors.initials: Q
authors.lastname: Yang
authors.affiliation: the Department of Physiology, John Hopkins University School of Medicine, Baltimore, Maryland 21205, and.
authors.forename: Dax
authors.initials: D
authors.lastname: Fu
authors.affiliation: the Physical Bioscience Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720.
authors.forename: Greg L
authors.initials: GL
authors.lastname: Hura
authors.affiliation: the Physical Bioscience Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720.
authors.forename: John A
authors.initials: JA
authors.lastname: Tainer
authors.affiliation: From the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602-7229, adams@bmb.uga.edu.
authors.forename: Michael W W
authors.initials: MW
authors.lastname: Adams
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D002462:Cell Membrane / Q000201:enzymology* / Q000235:genetics; D018360:Crystallography, X-Ray; D042967:Electron Transport Complex I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006864:Hydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics
chemical_list: D019843:Archaeal Proteins; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D042967:Electron Transport Complex I
keywords: Affinity Purification; Bioenergetics; Electron Transfer; Hydrogen; Hydrogenase; Membrane Enzyme; Respiration; Solubilization; Thermophile