ATP-Triggered Fe(CN)2CO Synthon Transfer from the Maturase HypCD to the Active Site of Apo-[NiFe]-Hydrogenase

[NiFe]-hydrogenases catalyze the reversible activation of H2 using a unique NiFe(CN)2CO metal site, which is assembled by a sophisticated multiprotein machinery. The [4Fe–4S] cluster-containing HypCD complex, which possesses an ATPase activity with a hitherto unknown function, serves as the hub for the assembly of the Fe(CN)2CO subfragment. HypCD is also thought to be responsible for the subsequent transfer of the iron fragment to the apo-form of the catalytic hydrogenase subunit, but the underlying mechanism has remained unexplored. Here, we performed a thorough spectroscopic characterization of different HypCD preparations using infrared, Mössbauer, and NRVS spectroscopy, revealing molecular details of the coordination of the Fe(CN)2CO fragment. Moreover, biochemical assays in combination with spectroscopy, AlphaFold structure predictions, protein–ligand docking calculations, and crosslinking MS deciphered unexpected mechanistic aspects of the ATP requirement of HypCD, which we found to actually trigger the transfer of the Fe(CN)2CO fragment to the apo-hydrogenase.

[镍铁]-氢化酶（[NiFe]-hydrogenases）可通过独特的NiFe(CN)₂CO金属位点催化氢气（H₂）的可逆活化，该位点的组装依赖一套复杂的多蛋白组装机器。含有[4Fe–4S]簇（[4Fe–4S] cluster）的HypCD复合物具备ATP酶活性，但其功能迄今尚未明确，该复合物是Fe(CN)₂CO亚片段组装的核心枢纽。此前研究推测HypCD还负责将该铁片段后续转移至催化氢化酶亚基的脱辅基形式（apo-form），但其背后的分子机制仍未被阐明。本研究通过红外光谱、穆斯堡尔光谱及核共振振动光谱（NRVS）对多种HypCD制剂开展了全面的光谱表征，揭示了Fe(CN)₂CO片段配位的分子细节。此外，结合生化实验与光谱分析、阿尔法折叠（AlphaFold）结构预测、蛋白质-配体对接计算以及交联质谱（crosslinking MS），本研究解析了HypCD的ATP需求相关的意外机制：我们发现ATP实际上会触发Fe(CN)₂CO片段向脱辅基氢化酶的转移。