The CO and CN− ligands to the active site Fe in [NiFe]-hydrogenase of Escherichia coli have different metabolic origins

The Fe atom in the bimetallic active site of [NiFe]-hydrogenases has one CO and two cyanide ligands. To determine their metabolic origin, [NiFe]-hydrogenase-2 was isolated from Escherichia coli grown in the presence of l-[ureido-13C]citrulline, purified and analyzed by infrared spectroscopy. The spectra indicate incorporation of 13C only into the cyanide ligands and not into the CO, showing that cyanide and CO have different metabolic origins. After growth of E. coli in the presence of 13CO only the CO ligand was labelled with 13C. Labelling did not result from an exchange of the intrinsic CO ligand with the exogenous CO.