title: [NiFe]-hydrogenase maturation in vitro: analysis of the roles of the HybG and HypD accessory proteins1.
abstract: [NiFe]-hydrogenases (Hyd) bind a nickel-iron-based cofactor. The Fe ion of the cofactor is bound by two cyanide ligands and a single carbon monoxide ligand. Minimally six accessory proteins (HypA-HypF) are necessary for NiFe(CN)2CO cofactor biosynthesis in Escherichia coli. It has been shown that the anaerobically purified HypC-HypD-HypE scaffold complex carries the Fe(CN)2CO moiety of this cofactor. In the present study, we have purified the HybG-HypDE complex and used it to successfully reconstitute in vitro active Hyd from E. coli. HybG is a homologue of HypC that is specifically required for the maturation of Hyd-2 and also functions in the maturation of Hyd-1 of E. coli. Maturation of active Hyd-1 and Hyd-2 could be demonstrated in extracts derived from HybG- and HypD-deficient E. coli strains by adding anaerobically purified HybG-HypDE complex. In vitro maturation was dependent on ATP, carbamoylphosphate, nickel and reducing conditions. Hydrogenase maturation was prevented when the purified HybG-HypDE complex used in the maturation assay lacked a bound Fe(CN)2CO moiety. These findings demonstrate that it is possible to isolate incompletely processed intermediates on the maturation pathway and to use these to activate apo-forms of [NiFe]-hydrogenase large subunits.
authors.affiliation: *Institute of Microbiology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle (Saale), Germany.
authors.forename: Basem
authors.initials: B
authors.lastname: Soboh
authors.affiliation: *Institute of Microbiology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle (Saale), Germany.
authors.forename: Ute
authors.initials: U
authors.lastname: Lindenstrauss
authors.affiliation: *Institute of Microbiology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle (Saale), Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Granich
authors.affiliation: *Institute of Microbiology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle (Saale), Germany.
authors.forename: Mahwish
authors.initials: M
authors.lastname: Javed
authors.affiliation: *Institute of Microbiology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle (Saale), Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Herzberg
authors.affiliation: *Institute of Microbiology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle (Saale), Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Thomas
authors.affiliation: †Freie Universität Berlin, Experimental Molecular Biophysics, Arnimalle 14, 14195 Berlin, Germany.
authors.forename: Sven T
authors.initials: ST
authors.lastname: Stripp
mesh_terms: D002248:Carbon Monoxide / Q000737:chemistry* / Q000378:metabolism; D003067:Coenzymes / Q000096:biosynthesis / Q000737:chemistry; D003486:Cyanides / Q000737:chemistry / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D006864:Hydrogenase / Q000737:chemistry / Q000378:metabolism*; D007501:Iron / Q000737:chemistry; D008024:Ligands; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism; D046912:Multiprotein Complexes / Q000737:chemistry / Q000302:isolation & purification; D009532:Nickel / Q000737:chemistry
chemical_list: D003067:Coenzymes; D003486:Cyanides; D029968:Escherichia coli Proteins; C508780:HybG protein, E coli; C491587:HypC protein, E coli; D008024:Ligands; D018832:Molecular Chaperones; D046912:Multiprotein Complexes; D009532:Nickel; D002248:Carbon Monoxide; D007501:Iron; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D010450:Endopeptidases; C119558:hydrogenase maturating endopeptidase HYBD