Deciphering the stepwise cellular assembly and integration of the [FeFe]-hydrogenase H-cluster

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The maturation of complex FeS-clusters in living cells usually requires a comparatively large number of helper proteins (maturases) that enable cofactor assembly in a sequential and well-coordinated manner. In case of the cofactor of [FeFe]-hydrogenases (H-cluster: [4Fe4S]-Cys-[2Fe2S](CO)3(CN-)2(CH2)2NH) the dedicated maturation system of the unique [2Fe2S]-sub-cluster (2FeH) consists of only three enzymes. The radical SAM proteins HydE and HydG provide the basic subparts (synthons), including the unusual ligand sphere of the Fe ions. The GTPase and scaffold protein HydF offers the proper environment for the assembly of a cofactor precursor (p2FeH) and determines the succession of assembly steps by specific consecutive interactions with HydE and HydG. While numerous aspects of synthon synthesis via HydG have been clarified, the steps of 2FeH assembly on HydF and the final steps of H-cluster integration into the non-maturated [FeFe]-hydrogenase protein are still not characterized in sufficient detail, leaving open several questions regarding the entire process. The list of unknowns includes the sequence of 2FeH assembly steps occurring on HydF, the nature of the corresponding cofactor intermediates and the exact coordination mode of the synthons within HydF. Additionally, the sequence of assembly steps that happen within the open H-cluster coordination site in apo-[FeFe]-hydrogenase after 2FeH-precursor transfer from mature HydF are yet to be understood. Concerning the latter aspect, we have already produced kinetic, IR-spectroscopic and electrochemical data in the framework of an exended variant study, which suggest certain basic amino acids to drive 2FeH cofactor incorporation into the apo-[FeFe]-hydrogenase. Moreover, five glycine positions appear to be required as hinge sites for the final restructuring process, which closes the H-cluster binding site after proper 2FeH incorporation. We aim at complementing these data by structural information from these variants prior and after 2FeH application. To understand the sequence of events required to assemble p2FeH on HydF, we will characterize intermediates of the structurally characterized HydF of Thermosipho melanesiensis and directed protein variants spectroscopically. Interestingly, the maturation system of green algal [FeFe]-hydrogenases is even more compact than what is known from bacteria, as the number of maturases is reduced to only two proteins, HydG and HydEF, the latter being a fusion protein between HydE and HydF. We plan to heterologously express and crystallize HydEF to understand the specifics of the fusion protein. Additionally, the characteristics of 2FeH precursor assembly on the HydF domain will be compared to those of T. melanesiensis HydF. Identified commonalities and differences will help us to fill the gaps in understanding the in vivo maturation process.