[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 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)(2)CO cofactor. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.