Towards a mechanistic understanding of the role of the iron-sulphur cluster-containing HypD protein in diatomic ligand biosynthesis of [NiFe]-hydrogenases

[NiFe]-hydrogenases are ancient iron-sulphur enzymes that have in their active site a bimetallic nickel-iron cofactor coordinated by four cysteinyl thiols. The cofactor is unusual in that the iron atom carries a carbon monoxide and two cyanides as diatomic ligands. Six conserved Hyp proteins are required to synthesize this NiFe(CN)₂CO moiety. The Fe(CN)₂CO portion of the cofactor is completed by the HypCDEF proteins and is inserted into the apo-catalytic subunit. Next, the nickel is inserted by HypA and HypB. The synthesis of the Fe(CN)₂CO group is not completely understood. The HypEF proteins generate the cyanide ligands from the precursor carbamoylphosphate. Recent evidence from our group suggests that the CO is generated from endogenous CO₂ bound to an iron atom that likely forms the basis of the cofactor. The key protein in Fe(CN)₂CO group synthesis is the iron-sulphur enzyme HypD. HypD functions as a scaffold with which the other Hyp proteins interact. Moreover, it is the only Hyp protein with a redox function. This research proposal, therefore, will focus primarily on elucidating the biochemical function of HypD in generating the Fe(CN)₂CO moiety. HypD functions together with a small ‚chaperone’-like protein HypC. HypC and HypD form a tight complex and as well as being responsible for recognition of the apo-catalytic subunit of the hydrogenase, HypC also binds iron bound with CO₂. Our current working hypothesis is that CO₂ bound by iron undergoes HypD-dependent reduction to an Fe(I)-CO intermediate to which the cyanide groups are transferred from the HypEF complex. We have recently demonstrated that HypD undergoes disulphide-thiol redox chemistry, explaining how CO₂ reduction might proceed. Therefore, we aim to determine the physiological electron donor to HypD, the mechanism by which HypD catalyzes reduction of CO₂ and how the HypCD complex transfers the Fe(CN)₂CO group into the active site of the hydrogenase