The complex between hydrogenase-maturation proteins HypC and HypD is an intermediate in the supply of cyanide to the active site iron of [NiFe]-hydrogenases.

Carbamoylphosphate has been shown to be the educt for the synthesis of
the CN ligands of the NiFe metal centre of hydrogenases from Escherichia
coli. In the absence of carbamoylphosphate, cells accumulate a complex of
two hydrogenase maturation proteins, namely HypC and HypD for the
synthesis of hydrogenase 3. A procedure for the purification of wild-type
HypD protein or of a biologically active derivative carrying the Strep-tagIIw
at the N terminus has been developed. HypD is a monomeric protein
possessing about 4 mol of iron per mol of protein. Electron paramagnetic
resonance (EPR) and Mo¿ssbauer spectroscopy demonstrated that the iron is
present as a diamagnetic [4Fe¿4S]2C cluster. The complex between HypC
and HypD can be cross-linked by a number of thiol and primary aminespecific
linkers. When HypD and HypC were overproduced side-by-side
with HypE, the HypC¿HypD complex contained substoichiometric
amounts of HypE whose proportion in the complex could be augmented
when HypF was also overproduced. HypE trapped in this complex could
be carbamoylated by protein HypF and after dehydration transferred the
cyano group to the HypC¿HypD part of the complex. Free HypC and
HypD were not cyanated by HypE-CN. An active HypC¿HypD complex
from anaerobic cells was inactivated by incubation with K3[Fe(CN)6] but
not with K4[Fe(CN)6]. The results suggest the existence of a dynamic
complex between the hydrogenase maturation proteins HypD, HypC,
HypE and HypF, which is the site of ligand biosynthesis and attachment to
the iron atom of the NiFe site in hydrogenase 3.