title: Structural differences of oxidized iron-sulfur and nickel-iron cofactors in O2-tolerant and O2-sensitive hydrogenases studied by X-ray absorption spectroscopy.
abstract: The class of [NiFe]-hydrogenases comprises oxygen-sensitive periplasmic (PH) and oxygen-tolerant membrane-bound (MBH) enzymes. For three PHs and four MBHs from six bacterial species, structural features of the nickel-iron active site of hydrogen turnover and of the iron-sulfur clusters functioning in electron transfer were determined using X-ray absorption spectroscopy (XAS). Fe-XAS indicated surplus oxidized iron and a lower number of ~2.7 Å Fe-Fe distances plus additional shorter and longer distances in the oxidized MBHs compared to the oxidized PHs. This supported a double-oxidized and modified proximal FeS cluster in all MBHs with an apparent trimer-plus-monomer arrangement of its four iron atoms, in agreement with crystal data showing a [4Fe3S] cluster instead of a [4Fe4S] cubane as in the PHs. Ni-XAS indicated coordination of the nickel by the thiol group sulfurs of four conserved cysteines and at least one iron-oxygen bond in both MBH and PH proteins. Structural differences of the oxidized inactive [NiFe] cofactor of MBHs in the Ni-B state compared to PHs in the Ni-A state included a ~0.05 Å longer Ni-O bond, a two times larger spread of the Ni-S bond lengths, and a ~0.1 Å shorter Ni-Fe distance. The modified proximal [4Fe3S] cluster, weaker binding of the Ni-Fe bridging oxygen species, and an altered localization of reduced oxygen species at the active site may each contribute to O2 tolerance.
authors.affiliation: Freie Universität Berlin, Institut für Experimentalphysik, 14195 Berlin, Germany.
authors.forename: Kajsa G V
authors.initials: KGV
authors.lastname: Sigfridsson
authors.affiliation: Freie Universität Berlin, Institut für Experimentalphysik, 14195 Berlin, Germany.
authors.forename: Nils
authors.initials: N
authors.lastname: Leidel
authors.affiliation: Freie Universität Berlin, Institut für Experimentalphysik, 14195 Berlin, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Sanganas
authors.affiliation: Freie Universität Berlin, Institut für Experimentalphysik, 14195 Berlin, Germany.
authors.forename: Petko
authors.initials: P
authors.lastname: Chernev
authors.affiliation: Humboldt-Universität zu Berlin, Mikrobiologie, 10115 Berlin, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Lenz
authors.affiliation: Ibaraki University, Department of Bioresource Science, Ibaraki 300-0393, Japan.
authors.forename: Ki-Seok
authors.initials: KS
authors.lastname: Yoon
authors.affiliation: Ibaraki University, Department of Bioresource Science, Ibaraki 300-0393, Japan.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Nishihara
authors.affiliation: University of Oxford, Department of Inorganic Chemistry, Oxford OX1 3QR, UK.
authors.forename: Alison
authors.initials: A
authors.lastname: Parkin
authors.affiliation: University of Oxford, Department of Inorganic Chemistry, Oxford OX1 3QR, UK.
authors.forename: Fraser A
authors.initials: FA
authors.lastname: Armstrong
authors.affiliation: CNRS Marseille, Unité de Bioénergétique et Ingénierie des Protéines, 13402 Marseille, France.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Dementin
authors.affiliation: CNRS Marseille, Unité de Bioénergétique et Ingénierie des Protéines, 13402 Marseille, France.
authors.forename: Marc
authors.initials: M
authors.lastname: Rousset
authors.affiliation: Instituto de Catálisis (CSIC), 28049 Madrid, Spain.
authors.forename: Antonio L
authors.initials: AL
authors.lastname: De Lacey
authors.affiliation: Freie Universität Berlin, Institut für Experimentalphysik, 14195 Berlin, Germany. Electronic address: michael.haumann@fu-berlin.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Haumann
mesh_terms: D001665:Binding Sites; D006864:Hydrogenase / Q000737:chemistry*; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D056928:X-Ray Absorption Spectroscopy / Q000379:methods*
chemical_list: D007506:Iron-Sulfur Proteins; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D010100:Oxygen
keywords: FeS cluster; O(2)-tolerance; X-ray absorption spectroscopy; [NiFe] active site; [NiFe]-hydrogenase