title: Reduction of unusual iron-sulfur clusters in the H2-sensing regulatory Ni-Fe hydrogenase from Ralstonia eutropha H16.
abstract: The regulatory Ni-Fe hydrogenase (RH) from Ralstonia eutropha functions as a hydrogen sensor. The RH consists of the large subunit HoxC housing the Ni-Fe active site and the small subunit HoxB containing Fe-S clusters. The heterolytic cleavage of H(2) at the Ni-Fe active site leads to the EPR-detectable Ni-C state of the protein. For the first time, the simultaneous but EPR-invisible reduction of Fe-S clusters during Ni-C state formation was demonstrated by changes in the UV-visible absorption spectrum as well as by shifts of the iron K-edge from x-ray absorption spectroscopy in the wild-type double dimeric RH(WT) [HoxBC](2) and in a monodimeric derivative designated RH(stop) lacking the C-terminal 55 amino acids of HoxB. According to the analysis of iron EXAFS spectra, the Fe-S clusters of HoxB pronouncedly differ from the three Fe-S clusters in the small subunits of crystallized standard Ni-Fe hydrogenases. Each HoxBC unit of RH(WT) seems to harbor two [2Fe-2S] clusters in addition to a 4Fe species, which may be a [4Fe-3S-3O] cluster. The additional 4Fe-cluster was absent in RH(stop). Reduction of Fe-S clusters in the hydrogen sensor RH may be a first step in the signal transduction chain, which involves complex formation between [HoxBC](2) and tetrameric HoxJ protein, leading to the expression of the energy converting Ni-Fe hydrogenases in R. eutropha.
authors.affiliation: Humboldt-Universität zu Berlin, Institut für Biologie/Mikrobiologie, Berlin, Germany.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Buhrke
authors.forename: Simone
authors.initials: S
authors.lastname: Löscher
authors.forename: Oliver
authors.initials: O
authors.lastname: Lenz
authors.forename: Eberhard
authors.initials: E
authors.lastname: Schlodder
authors.forename: Ingo
authors.initials: I
authors.lastname: Zebger
authors.forename: Lars K
authors.initials: LK
authors.lastname: Andersen
authors.forename: Peter
authors.initials: P
authors.lastname: Hildebrandt
authors.forename: Wolfram
authors.initials: W
authors.lastname: Meyer-Klaucke
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: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D020118:Cupriavidus necator / Q000201:enzymology* / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D006864:Hydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010084:Oxidation-Reduction; D021122:Protein Subunits; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D017550:Spectroscopy, Fourier Transform Infrared; D013057:Spectrum Analysis; D014965:X-Rays
chemical_list: D021122:Protein Subunits; C043296:nickel-iron hydrogenase; D006864:Hydrogenase