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

FEBS Lett. 2007 Jul 10;581(17):3322-6. doi: 10.1016/j.febslet.2007.06.027. Epub 2007 Jun 21.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Carbamyl Phosphate / metabolism*
  • Carbon Dioxide / metabolism
  • Carbon Isotopes / pharmacokinetics
  • Carbon Monoxide / metabolism*
  • Citrulline / pharmacokinetics
  • Cloning, Molecular
  • Cupriavidus necator / enzymology*
  • Cupriavidus necator / metabolism
  • Cyanides / metabolism*
  • Hydrogenase / chemistry
  • Hydrogenase / genetics
  • Hydrogenase / metabolism*
  • Protein Subunits / metabolism
  • Protein Subunits / physiology

Substances

  • Carbon Isotopes
  • Cyanides
  • Protein Subunits
  • Carbon Dioxide
  • Citrulline
  • Carbamyl Phosphate
  • Carbon Monoxide
  • nickel-iron hydrogenase
  • Hydrogenase