The role of the iron-sulphur cluster-containing HypD ATPase and its interplay with other Hyp proteins during the maturation of NiFe-hydrogenases

. In this funding period, we will characterize the ATPase activity of HypD in more detail. We will determine which amino acids and motifs are required for the ATPase activity of HypD. We will also determine whether the interaction of HypD with HypC/HybG influences ATP hydrolysis. The fact that ATP is hydrolyzed by HypD is congruent with our working hypothesis that the enzyme reduces CO₂ (E^o’ = ca.-530 mV) or possibly a carboxylate group and using the ATPase assay we have developed, and we will attempt to demonstrate this activity. Further experiments will focus on the order-of-addition of the diatomic ligands, the transfer of the Fe-(CN)₂CO group by HypC/HybG to the large subunit and the identification of further interaction partners of both HypD and HypC to provide insight into the in vivo donors of both the Fe ion as well as the precursor metabolite of the CO ligand.