title: 17O ENDOR detection of a solvent-derived Ni-(OH(x))-Fe bridge that is lost upon activation of the hydrogenase from Desulfovibrio gigas.
abstract: Crystallographic studies of the hydrogenases (Hases) from Desulfovibrio gigas (Dg) and Desulfovibrio vulgaris Miyazaki (DvM) have revealed heterodinuclear nickel-iron active centers in both enzymes. The structures, which represent the as-isolated (unready) Ni-A (S = (1)/(2)) enzyme state, disclose a nonprotein ligand (labeled as X) bridging the two metals. The bridging atom was suggested to be an oxygenic (O(2)(-) or OH(-)) species in Dg Hase and an inorganic sulfide in DvM Hase. To determine the nature and chemical characteristics of the Ni-X-Fe bridging ligand in Dg Hase, we have performed 35 GHz CW (17)O ENDOR measurements on the Ni-A form of the enzyme, exchanged into H(2)(17)O, on the active Ni-C (S = (1)/(2)) form prepared by H(2)-reduction of Ni-A in H(2)(17)O, and also on Ni-A formed by reoxidation of Ni-C in H(2)(17)O. In the native state of the protein (Ni-A), the bridging ligand does not exchange with the H(2)(17)O solvent. However, after a reduction/reoxidation cycle (Ni-A --> Ni-C --> Ni-A), an (17)O label is introduced at the active site, as seen by ENDOR. Detailed analysis of a 2-D field-frequency plot of ENDOR spectra taken across the EPR envelope of Ni-A((17)O) shows that the incorporated (17)O has a roughly axial hyperfine tensor, A((17)O) approximately [5, 7, 20] MHz, discloses its orientation relative to the g tensor, and also yields an estimate of the quadrupole tensor. The substantial isotropic component (a(iso)((17)O) approximately 11 MHz) of the hyperfine interaction indicates that a solvent-derived (17)O is indeed a ligand to Ni and thus that the bridging ligand X in the Ni-A state of Dg Hase is indeed an oxygenic (O(2)(-) or OH(-)) species; comparison with earlier EPR results by others indicates that the same holds for Ni-B. The small (57)Fe hyperfine coupling seen previously for Ni-A (A((57)Fe) approximately 0.9 MHz) is now shown to persist in Ni-C, A((57)Fe) approximately 0.8 MHz. However, the (17)O signal is lost upon reductive activation to the Ni-C state; reoxidation to Ni-A leads to the reappearance of the signal. Consideration of the electronic structure of the EPR-active states of the dinuclear center leads us to suggest that the oxygenic bridge in Ni-A(B) is lost in Ni-C and is re-formed from solvent upon reoxidation to Ni-A. This implies that the reductive activation to Ni-C opens Ni/Fe coordination sites which may play a central role in the enzyme's activity.
authors.affiliation: Departamento de Quimica and Centro de Química Fina e Biotecnologia, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2825-114 Monte de Caparica, Portugal.
authors.forename: Marta
authors.initials: M
authors.lastname: Carepo
authors.forename: David L
authors.initials: DL
authors.lastname: Tierney
authors.forename: Carlos D
authors.initials: CD
authors.lastname: Brondino
authors.forename: Tran Chin
authors.initials: TC
authors.lastname: Yang
authors.forename: Ana
authors.initials: A
authors.lastname: Pamplona
authors.forename: Joshua
authors.initials: J
authors.lastname: Telser
authors.forename: Isabel
authors.initials: I
authors.lastname: Moura
authors.forename: José J G
authors.initials: JJ
authors.lastname: Moura
authors.forename: Brian M
authors.initials: BM
authors.lastname: Hoffman
mesh_terms: D001665:Binding Sites; D003901:Desulfovibrio / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D006864:Hydrogenase / Q000737:chemistry* / Q000378:metabolism*; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009532:Nickel / Q000737:chemistry / Q000378:metabolism; D010103:Oxygen Isotopes
chemical_list: D010103:Oxygen Isotopes; D009532:Nickel; D007501:Iron; C043296:nickel-iron hydrogenase; D006864:Hydrogenase