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

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-C-13] citrulline and (NaHCO3)-C-13. Infrared spectroscopy of purified hydrogenase provided direct evidence that only the cyanide ligands, but not the CO ligand, originate from CO2 and carbamoylphosphate. Incorporation of label from (CO)-C-13 exclusively into the carbonyl ligand indicates that free CO is a possible precursor in carbonyl ligand biosynthesis. (C) 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.