The HypC and HypD maturases are required for the biosynthesis of the Fe(CN)2CO
cofactor in the large subunit of [NiFe]-hydrogenases. Using infrared
spectroscopy we demonstrate that an anaerobically purified, Strep-tagged HypCD
complex from Escherichia coli exhibits absorption bands characteristic of
diatomic CO and CN− ligands as well as CO2. Metal and sulphide analyses
revealed that along with the [4Fe–4S]2+ cluster in HypD, the complex has two
additional oxygen-labile Fe ions. We prove that HypD cysteine 41 is required
for the coordination of all three ligands. These findings suggest that the
HypCD complex carries minimally the Fe(CN)2CO cofactor
