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)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

The [NiFe]-hydrogenase accessory chaperones HypC and HybG of Escherichia coli are iron- and carbon dioxide-binding proteins.

[NiFe]-hydrogenase accessory proteins HypC and HypD form a complex that binds a Fe–(CN)2CO moiety and CO2. In this study two HypC homologues from Escherichia coli were purified under strictly anaerobic conditions and both contained sub-stoichiometric amounts of iron (approx. 0.3 mol Fe/mol HypC). Infrared spectroscopic analysis identified a signature at 2337 cm−1 indicating bound CO2. Aerobically isolated HypC lacked both Fe and CO2. Exchange of either of the highly conserved amino acid residues Cys2 or His51 abolished both Fe- and CO2-binding. Our results suggest that HypC delivers CO2 bound directly to Fe for reduction to CO by HypD

HypD Is the Scaffold Protein for Fe-(CN)₂CO Cofactor Assembly in [NiFe]-Hydrogenase Maturation

[NiFe]-hydrogenases bind a NiFe–(CN)₂CO cofactor in their catalytic large subunit. The iron–sulfur protein HypD and the small accessory protein HypC play a central role in the generation of the CO and CN^– ligands. Infrared spectroscopy identified signatures on an anaerobically isolated HypCD complex that are reminiscent of those in the hydrogenase active site, suggesting that this complex is the assembly site of the Fe-(CN)₂CO moiety of the cofactor prior to its transfer to the hydrogenase large subunit. Here, we report that HypD isolated in the absence of HypC shows infrared bands at 1956 cm^–1, 2072 cm^–1, and 2092 cm^–1 that can be assigned to CO, CN¹, and CN², respectively, and which are indistinguishable from those observed for the HypCD complex. HypC could not be isolated with CO or CN^– ligand contribution. Treatment of HypD with EDTA led to the concomitant loss of Fe and the CO and CN^– signatures, while oxidation by H₂O₂ resulted in a positive shift of the CO and CN^– bands by 35 cm^–1 and 20 cm^–1, respectively, indicative of the ferrous iron as an immediate ligation site for the diatomic ligands. Analysis of HypD amino acid variants identified cysteines 41, 69, and 72 to be essential for maturation of the cofactor. We propose a refined model for the ligation of Fe-(CN)₂CO to HypD and the role of HypC in [NiFe]-hydrogenase maturation