title: Engineering the respiratory membrane-bound hydrogenase of the hyperthermophilic archaeon Pyrococcus furiosus and characterization of the catalytically active cytoplasmic subcomplex.
abstract: The archaeon Pyrococcus furiosus grows optimally at 100°C by converting carbohydrates to acetate, carbon dioxide and hydrogen gas (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 classified as a Group 4 hydrogenase and is encoded by a 14-gene operon that contains hydrogenase and Na(+)/H(+) antiporter modules. Herein a His-tagged 4-subunit cytoplasmic subcomplex of MBH (C-MBH) was engineered and expressed in P. furiosus by differential transcription of the MBH operon. It was purified under anaerobic conditions by affinity chromatography without detergent. Purified C-MBH had a Fe : Ni ratio of 14 : 1, similar to the predicted value of 13 : 1. The O2 sensitivities of C-MBH and the 14-subunit membrane-bound version were similar (half-lives of ∼15 h in air), but C-MBH was more thermolabile (half-lives at 90°C of 8 and 25 h, respectively). C-MBH evolved H2 with the physiological electron donor, reduced ferredoxin, optimally at 60°C. This is the first report of the engineering and characterization of a soluble catalytically active subcomplex of a membrane-bound respiratory hydrogenase.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602-7229, USA.
authors.forename: Patrick M
authors.initials: PM
authors.lastname: McTernan
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602-7229, USA.
authors.forename: Sanjeev K
authors.initials: SK
authors.lastname: Chandrayan
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602-7229, USA.
authors.forename: Chang-Hao
authors.initials: CH
authors.lastname: Wu
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602-7229, USA.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Vaccaro
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602-7229, USA.
authors.forename: W Andrew
authors.initials: WA
authors.lastname: Lancaster
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602-7229, USA 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*; D006864:Hydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics
chemical_list: D019843:Archaeal Proteins; D008565:Membrane Proteins; D021122:Protein Subunits; D006864:Hydrogenase
keywords: Archaea; Differential expression; Hydrogenase; Membrane protein