title: Spectroscopic insights into the oxygen-tolerant membrane-associated [NiFe] hydrogenase of Ralstonia eutropha H16.
abstract: This study provides the first spectroscopic characterization of the membrane-bound oxygen-tolerant [NiFe] hydrogenase (MBH) from Ralstonia eutropha H16 in its natural environment, the cytoplasmic membrane. The H2-converting MBH is composed of a large subunit, harboring the [NiFe] active site, and a small subunit, capable in coordinating one [3Fe4S] and two [4Fe4S] clusters. The hydrogenase dimer is electronically connected to a membrane-integral cytochrome b. EPR and Fourier transform infrared spectroscopy revealed a strong similarity of the MBH active site with known [NiFe] centers from strictly anaerobic hydrogenases. Most redox states characteristic for anaerobic [NiFe] hydrogenases were identified except for one remarkable difference. The formation of the oxygen-inhibited Niu-A state was never observed. Furthermore, EPR data showed the presence of an additional paramagnetic center at high redox potential (+290 mV), which couples magnetically to the [3Fe4S] center and indicates a structural and/or redox modification at or near the proximal [4Fe4S] cluster. Additionally, significant differences regarding the magnetic coupling between the Nia-C state and [4Fe4S] clusters were observed in the reduced form of the MBH. The spectroscopic properties are discussed with regard to the unusual oxygen tolerance of this hydrogenase and in comparison with those of the solubilized, dimeric form of the MBH.
authors.affiliation: From the Institut für Chemie, Technische Universität Berlin, PC14, Strasse des 17. Juni 135, D-10623 Berlin.
authors.forename: Miguel
authors.initials: M
authors.lastname: Saggu
authors.affiliation: From the Institut für Chemie, Technische Universität Berlin, PC14, Strasse des 17. Juni 135, D-10623 Berlin. Electronic address: ingo.zebger@tu-berlin.de.
authors.forename: Ingo
authors.initials: I
authors.lastname: Zebger
authors.affiliation: Institute of Biology, Department of Microbiology, Humboldt-Universität zu Berlin, Chausseestrasse 117, D-10115 Berlin, Germany.
authors.forename: Marcus
authors.initials: M
authors.lastname: Ludwig
authors.affiliation: Institute of Biology, Department of Microbiology, Humboldt-Universität zu Berlin, Chausseestrasse 117, D-10115 Berlin, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Lenz
authors.affiliation: Institute of Biology, Department of Microbiology, Humboldt-Universität zu Berlin, Chausseestrasse 117, D-10115 Berlin, Germany.
authors.forename: Bärbel
authors.initials: B
authors.lastname: Friedrich
authors.affiliation: From the Institut für Chemie, Technische Universität Berlin, PC14, Strasse des 17. Juni 135, D-10623 Berlin.
authors.forename: Peter
authors.initials: P
authors.lastname: Hildebrandt
authors.affiliation: From the Institut für Chemie, Technische Universität Berlin, PC14, Strasse des 17. Juni 135, D-10623 Berlin. Electronic address: f.lendzian@tu-berlin.de.
authors.forename: Friedhelm
authors.initials: F
authors.lastname: Lendzian
mesh_terms: D002384:Catalysis; D002392:Catechin / Q000378:metabolism; D020118:Cupriavidus necator / Q000201:enzymology*; D003593:Cytoplasm / Q000378:metabolism; D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy; D006864:Hydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007501:Iron / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009532:Nickel / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D010957:Plasmids; D012995:Solubility; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D008565:Membrane Proteins; D009532:Nickel; D002392:Catechin; D007501:Iron; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D010100:Oxygen