[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 molFe/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.
Keywords: Carbon dioxide; Hydrogenase; Infrared spectroscopy; Iron; Maturation; Metalloprotein.
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