[NiFe]‐hydrogenase maturation: Isolation of a HypC–HypD complex carrying diatomic CO and CN⁻ ligands

The HypC and HypD maturases are required for the biosynthesis of the Fe(CN)(2)CO 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 CO(2). 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)(2)CO cofactor.