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

复杂的FeS簇在活细胞中的成熟通常需要相对大量的辅助蛋白（成熟酶），其能够以顺序和良好协调的方式进行辅因子组装。在[FeFe]-氢化酶（H-簇：[4Fe 4S]-Cys-[2Fe 2S]（CO）3（CN-）2（CH 2）2NH）的辅因子的情况下，独特的[2Fe 2S]-亚簇（2FeH）的专用成熟系统仅由三种酶组成。自由基SAM蛋白质HydE和HydG提供了基本的子部分（离子），包括不寻常的配体领域的铁离子。GTdE和支架蛋白HydF为辅因子前体（p2 FeH）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确定了组装步骤的顺序。虽然通过HydG的合成子合成的许多方面已经得到澄清，HydF上的2FeH组装步骤和H-簇整合到未成熟的[FeFe]-氢化酶蛋白质中的最后步骤仍然没有足够详细的特征，留下了关于整个过程的几个问题。未知数的列表包括2FeH组装步骤发生在HydF上的顺序，相应的辅因子中间体的性质和HydF内的配体的确切配位模式。此外，在2FeH-前体从成熟HydF转移后，在apo-[FeFe]-氢化酶中的开放H-簇配位位点内发生的组装步骤的顺序尚未被理解。关于后一个方面，我们已经产生了动力学，红外光谱和电化学数据的框架内的一个扩展的变体研究，这表明某些碱性氨基酸驱动2FeH辅因子纳入载脂蛋白-[FeFe]-氢化酶。此外，五个甘氨酸位置似乎需要作为铰链网站的最终重组过程中，关闭H-簇结合位点后，适当的2FeH掺入。我们的目标是通过这些变体的结构信息来补充这些数据，在2FeH应用之前和之后。为了了解组装p2 FeH对HydF所需的事件序列，我们将表征中间体的结构特征HydF的Thermosipho melanesiensis和定向蛋白质变体光谱。有趣的是，绿色藻类[FeFe]-氢化酶的成熟系统甚至比已知的来自细菌的成熟系统更紧凑，因为成熟酶的数量减少到只有两种蛋白质，HydG和HydEF，后者是HydE和HydF之间的融合蛋白。我们计划异源表达和结晶HydEF以了解融合蛋白的特性。此外，2FeH前体组装在HydF结构域上的特征将与T.墨角藓确定的共性和差异将有助于我们填补理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.