title: Carbamoylphosphate serves as the source of CN(-), but not of the intrinsic CO in the active site of the regulatory [NiFe]-hydrogenase from Ralstonia eutropha.
abstract: Within the catalytic centre of [NiFe]-hydrogenases one carbonyl and two cyanide ligands are covalently attached to the iron. To identify the metabolic origins of these ligands, the regulatory [NiFe] hydrogenase in conjunction with the indigenous Hyp maturation proteins of Ralstonia eutropha H16 were heterologously overproduced in E. coli grown in the presence of L-[ureido-(13)C] citrulline and NaH(13)CO(3). Infrared spectroscopy of purified hydrogenase provided direct evidence that only the cyanide ligands, but not the CO ligand, originate from CO(2) and carbamoylphosphate. Incorporation of label from (13)CO exclusively into the carbonyl ligand indicates that free CO is a possible precursor in carbonyl ligand biosynthesis.
authors.affiliation: Humboldt-Universität zu Berlin, Institut für Biologie/Mikrobiologie, Chausseestrasse 117, Berlin, Germany. oliver.lenz@rz.hu-berlin.de
authors.forename: Oliver
authors.initials: O
authors.lastname: Lenz
authors.forename: Ingo
authors.initials: I
authors.lastname: Zebger
authors.forename: Josta
authors.initials: J
authors.lastname: Hamann
authors.forename: Peter
authors.initials: P
authors.lastname: Hildebrandt
authors.forename: Bärbel
authors.initials: B
authors.lastname: Friedrich
mesh_terms: D001665:Binding Sites; D002221:Carbamyl Phosphate / Q000378:metabolism*; D002245:Carbon Dioxide / Q000378:metabolism; D002247:Carbon Isotopes / Q000493:pharmacokinetics; D002248:Carbon Monoxide / Q000378:metabolism*; D002956:Citrulline / Q000493:pharmacokinetics; D003001:Cloning, Molecular; D020118:Cupriavidus necator / Q000201:enzymology* / Q000378:metabolism; D003486:Cyanides / Q000378:metabolism*; D006864:Hydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism / Q000502:physiology
chemical_list: D002247:Carbon Isotopes; D003486:Cyanides; D021122:Protein Subunits; D002245:Carbon Dioxide; D002956:Citrulline; D002221:Carbamyl Phosphate; D002248:Carbon Monoxide; C043296:nickel-iron hydrogenase; D006864:Hydrogenase