[FeFe] hydrogenases have attracted extensive attention in the field of renewable energy research because of their remarkable efficiency for H2 gas production. H2 formation is catalyzed by a biologically unique hexanuclear iron cofactor denoted the H-cluster. The assembly of this cofactor requires a dedicated maturation machinery including HydF, a multidomain [4Fe4S] cluster protein with GTPase activity. HydF is responsible for harboring and delivering a precatalyst to the apo-hydrogenase, but the details of this process are not well understood. Here, we utilize gas-phase electrophoretic macromolecule analysis to show that a HydF dimer forms a transient interaction complex with the hydrogenase and that the formation of this complex depends on the cofactor content on HydF. Moreover, Fourier transform infrared, electron paramagnetic resonance, and UV-visible spectroscopy studies of mutants of HydF show that the isolated iron-sulfur cluster domain retains the capacity for binding the precatalyst in a reversible fashion and is capable of activating apo-hydrogenase in in vitro assays. These results demonstrate the central role of the iron-sulfur cluster domain of HydF in the final stages of H-cluster assembly, i.e. in binding and delivering the precatalyst.

中文翻译：

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成熟酶HydF通过瞬时的辅因子依赖性相互作用实现[FeFe]氢化酶组装。

[FeFe]氢化酶因其生产氢气的出色效率而在可再生能源研究领域引起了广泛关注。H2的形成由称为H簇的生物学上独特的六核铁辅因子催化。该辅因子的组装需要专用的成熟机制，包括HydF，一种具有GTPase活性的多域[4Fe4S]簇蛋白。HydF负责保留和传递载脂蛋白加氢酶的前催化剂，但该过程的细节尚未完全了解。在这里，我们利用气相电泳大分子分析显示，HydF二聚体与氢化酶形成了一个短暂的相互作用复合物，并且该复合物的形成取决于HydF上辅因子的含量。此外，傅立叶变换红外，电子顺磁共振，HydF突变体的紫外和可见光谱研究表明，分离的铁硫簇结构域以可逆的方式保留了结合前催化剂的能力，并且能够在体外测定中激活脱辅基加氢酶。这些结果证明了HydF的铁-硫簇结构域在H-簇组装的最后阶段，即在结合和递送预催化剂中的核心作用。