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

活细胞中复杂fes簇的成熟通常需要相对大量的辅助蛋白（成熟酶），这些辅助蛋白能够以顺序和良好协调的方式组装辅因子。以[FeFe]-氢化酶的辅助因子(h -簇：[4Fe4S]- cys -[2Fe2S](CO)3（CN-)2(CH2)2NH）为例，独特的[2Fe2S]-亚簇（2FeH）的专用成熟系统仅由三种酶组成。自由基SAM蛋白HydE和HydG提供基本子（合子），包括不寻常的Fe离子配体球。GTPase和支架蛋白HydF为辅助因子前体（p2FeH）的组装提供了合适的环境，并通过与HydE和HydG的特定连续相互作用决定了组装步骤的连续性。虽然通过HydG合成的许多方面已经得到澄清，但在HydF上组装2FeH的步骤以及h簇整合到未成熟的[FeFe]-氢化酶蛋白的最后步骤仍然没有得到足够详细的表征，留下了关于整个过程的几个问题。未知列表包括发生在HydF上的2FeH组装步骤的顺序，相应的辅因子中间体的性质以及HydF内合成子的确切协调模式。此外，在成熟的HydF转移2feh前体后，apo-[FeFe]-氢化酶的开放h簇配位位点内发生的组装步骤序列尚不清楚。关于后一个方面，我们已经在一个扩展的变异研究框架中产生了动力学、红外光谱和电化学数据，这些数据表明某些碱性氨基酸驱动2FeH辅因子结合到载铁-[FeFe]-氢化酶中。此外，5个甘氨酸位点似乎需要作为最终重组过程的铰链位点，在适当的2FeH结合后关闭h簇结合位点。我们的目标是通过在2FeH应用之前和之后从这些变体中获得的结构信息来补充这些数据。为了了解在HydF上组装p2FeH所需的事件序列，我们将对热sipho melanesiensis结构表征的HydF和定向蛋白变体的中间产物进行光谱表征。有趣的是，绿藻[FeFe]-氢化酶的成熟系统甚至比已知的细菌更紧凑，因为成熟酶的数量减少到只有两种蛋白质，HydG和HydEF，后者是海德和HydF之间的融合蛋白。我们计划异种表达和结晶HydEF以了解融合蛋白的特性。此外，将2FeH前体在HydF结构域上的组装特性与T. melanesiensis HydF进行比较。确定的共性和差异将有助于我们填补了解体内成熟过程的空白。

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.