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

• Applicant: Professor Dr. Gary Sawers
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2016 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 310984010

[NiFe]-hydrogenases are iron-sulphur (FeS) enzymes that have a bimetallic NiFe(CN)2CO cofactor coordinated by four cysteinyl thiols in the active site of the large subunit. The cofactor is synthesized by six conserved Hyp proteins. Synthesis of the Fe(CN)2CO portion of the cofactor is completed on a HypCDEF protein scaffold and is then inserted into a precursor of the large subunit. HypD forms the core of the scaffold complex and together with HypC coordinates the Fe(CN)2CO group via two conserved cysteinyl thiols; one from HypD (C41) and one from HypC (C2). The cyanide ligands are generated by the combined actions of the carbamoyltransferase HypF and the dehydratase HypE. The synthesis route of the CO ligand to the Fe on the cofactor is still unresolved, as is when it is added, but our working hypothesis is that this originates from CO2 previously shown to be coordinated to the Fe. HypD has a [4Fe-4S] cluster and in the last funding period we have determined the redox potential of the cluster to be Eo’ = -260 mV. We have also shown that two conserved Cys residues (C69 and C72) undergo disulfide-thiol exchange in two consecutive one-electron and -proton transfer steps. Moreover, we showed that HypD has an ATPase activity (ADP is formed), which depends on Cys41. While trying to identify the electron donor to HypD’s FeS cluster, we could exclude ferredoxin in this capacity and we are currently analyzing whether one of the two flavodoxins found in Escherichia coli functions in this role. Using protein-protein interaction studies combined with native mass spectrometry, we showed that only HybG, a paralogue of HypC, interacts with the large subunit precursor. HypD does not enter into a ternary complex with HybG and the large subunit precursor. Moreover, HybG only interacts with the precursor and not the C-terminally processed, mature large subunit. Thus, HypC/HybG enters into a complex either with HypD or with the large subunit precursor and functions both in the construction of the Fe(CN)2CO group and its final delivery to the target protein via a thiol transferase reaction. In the next 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 (Eo’ = ca.-530 mV) or possibly a carboxylate group and using the ATPase assay we have developed, 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 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.

• DFG Programme: Priority Programmes
• Subproject of: SPP 1927: Iron-Sulfur for Life