title: A universal scaffold for synthesis of the Fe(CN)2(CO) moiety of [NiFe] hydrogenase.
abstract: Hydrogen-cycling [NiFe] hydrogenases harbor a dinuclear catalytic center composed of nickel and iron ions, which are coordinated by four cysteine residues. Three unusual diatomic ligands in the form of two cyanides (CN(-)) and one carbon monoxide (CO) are bound to the iron and apparently account for the complexity of the cofactor assembly process, which involves the function of at least six auxiliary proteins, designated HypA, -B, -C, -D, -E, and -F. It has been demonstrated previously that the HypC, -D, -E, and -F proteins participate in cyanide synthesis and transfer. Here, we show by infrared spectroscopic analysis that the purified HypCD complexes from Ralstonia eutropha and Escherichia coli carry in addition to both cyanides the CO ligand. We present experimental evidence that in vivo the attachment of the CN(-) ligands is a prerequisite for subsequent CO binding. With the aid of genetic engineering and subsequent mutant analysis, the functional role of conserved cysteine residues in HypD from R. eutropha was investigated. Our results demonstrate that the HypCD complex serves as a scaffold for the assembly of the Fe(CN)(2)(CO) entity of [NiFe] hydrogenase.
authors.affiliation: Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Chausseestrasse 117,10115 Berlin, Germany.
authors.forename: Ingmar
authors.initials: I
authors.lastname: Bürstel
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Siebert
authors.forename: Gordon
authors.initials: G
authors.lastname: Winter
authors.forename: Philipp
authors.initials: P
authors.lastname: Hummel
authors.forename: Ingo
authors.initials: I
authors.lastname: Zebger
authors.forename: Bärbel
authors.initials: B
authors.lastname: Friedrich
authors.forename: Oliver
authors.initials: O
authors.lastname: Lenz
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D002248:Carbon Monoxide / Q000737:chemistry; D020134:Catalytic Domain; D020118:Cupriavidus necator / Q000378:metabolism; D003486:Cyanides / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods; D006864:Hydrogenase / Q000737:chemistry*; D007477:Ions; D007501:Iron / Q000737:chemistry; D008024:Ligands; D008670:Metals / Q000737:chemistry; D011506:Proteins / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D003486:Cyanides; C108874:HypC protein, Bacteria; C108875:HypD protein, Bacteria; D007477:Ions; D008024:Ligands; D008670:Metals; D011506:Proteins; D002248:Carbon Monoxide; D007501:Iron; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D003545:Cysteine