title: Bias from H2 cleavage to production and coordination changes at the Ni-Fe active site in the NAD+-reducing hydrogenase from Ralstonia eutropha.
abstract: The soluble NAD+-reducing Ni-Fe hydrogenase (SH) from Ralstonia eutropha H16 is remarkable because it cleaves hydrogen in the presence of dioxygen at a unique Ni-Fe active site (Burgdorf et al. (2005) J. Am. Chem. Soc. 127, 576). By X-ray absorption (XAS), FTIR, and EPR spectroscopy, we monitored the structure and oxidation state of its metal centers during H2 turnover. In NADH-activated protein, a change occurred from the (CN)O2Ni(II)(mu-S)2Fe(II)(CN)3(CO) site dominant in the wild-type SH to a standard-like S2Ni(II)(mu-S)2Fe(II)(CN)2(CO) site as the prevailing species in a specific mutant protein, HoxH-H16L. The wild-type SH primarily was active in H2 cleavage. The nonstandard reaction mechanism does not involve stable EPR-detectable trivalent Ni oxidation states, namely, the Ni-A,B,C states as observed in standard hydrogenases. In the HoxH-mutant protein H16L, H2 oxidation was impaired, but H2 production occurred via a stable Ni-C state (Ni(III)-H(-)-Fe(II)), suggesting a reaction sequence similar to that of standard hydrogenases. It is proposed that reductive activation by NADH of both wild-type and H16L proteins causes the release of an oxygen species from Ni and is initiated by electron transfer from a [2Fe-2S] cluster in the HoxU subunit that at first becomes reduced by electrons from NADH. Electrons derived from H2 cleavage, on the other hand, are transferred to NAD+ via a different pathway involving a [4Fe-4S] cluster in HoxY, which is reducible only in wild-type SH but not in the H16L variant.
authors.affiliation: Freie Universität Berlin, Institut für Experimentalphysik, Arnimallee 14, D-14195 Berlin, Germany.
authors.forename: Simone
authors.initials: S
authors.lastname: Löscher
authors.forename: Tanja
authors.initials: T
authors.lastname: Burgdorf
authors.forename: Ingo
authors.initials: I
authors.lastname: Zebger
authors.forename: Peter
authors.initials: P
authors.lastname: Hildebrandt
authors.forename: Holger
authors.initials: H
authors.lastname: Dau
authors.forename: Bärbel
authors.initials: B
authors.lastname: Friedrich
authors.forename: Michael
authors.initials: M
authors.lastname: Haumann
mesh_terms: D001665:Binding Sites; D020118:Cupriavidus necator / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D006859:Hydrogen / Q000378:metabolism*; D007501:Iron / Q000378:metabolism*; D008954:Models, Biological; D050505:Mutant Proteins / Q000378:metabolism; D009532:Nickel / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared; D014732:Vibration
chemical_list: D050505:Mutant Proteins; D009532:Nickel; D006859:Hydrogen; D007501:Iron; D010088:Oxidoreductases; C021376:hydrogen dehydrogenase