The [4Fe-4S] cluster containing scaffold complex HypCD is the central construction site for the assembly of the [Fe](CN)2CO cofactor precursor of [NiFe]-hydrogenase. While the importance of the HypCD complex is well established, not much is known about the mechanism by which the CN− and CO ligands are transferred and attached to the iron ion. We report an efficient expression and purification system producing the HypCD complex from E. coli with complete metal content. This enabled in-depth spectroscopic characterizations. The results obtained by EPR and Mössbauer spectroscopy demonstrate that the [Fe](CN)2CO cofactor and the [4Fe-4S] cluster of the HypCD complex are redox active. The data indicate a potential-dependent interconversion of the [Fe]2+/3+ and [4Fe-4S]2+/+ couple, respectively. Moreover, ATR FTIR spectroscopy reveals potential-dependent disulfide formation, which hints at an electron confurcation step between the metal centers. MicroScale thermophoresis indicates preferable binding between the HypCD complex and its in vivo interaction partner HypE under reducing conditions. Together, these results provide comprehensive evidence for an electron inventory fit to drive multi-electron redox reactions required for the assembly of the CN− and CO ligands on the scaffold complex HypCD.

中文翻译：

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[NiFe]-氢化酶辅因子组装中必不可少的铁硫支架复合物 HypCD 的电子库存

含有支架复合物 HypCD 的 [4Fe-4S] 簇是组装 [NiFe]-氢化酶的 [Fe](CN)2CO 辅因子前体的中心构建位点。虽然 HypCD 复合物的重要性已得到充分证实，但对 CN- 和 CO 配体转移并附着到铁离子的机制知之甚少。我们报告了一种有效的表达和纯化系统，可从大肠杆菌中产生具有完整金属含量的 HypCD 复合物。这实现了深入的光谱表征。通过 EPR 和穆斯堡尔光谱获得的结果表明，HypCD 复合物的 [Fe](CN)2CO 辅因子和 [4Fe-4S] 簇具有氧化还原活性。数据分别表明 [Fe]2+/3+ 和 [4Fe-4S]2+/+ 对的电位依赖性相互转换。而且，ATR FTIR 光谱揭示了电位依赖性二硫化物的形成，这暗示了金属中心之间的电子分叉步骤。MicroScale 热泳表明 HypCD 复合物与其体内相互作用伙伴 HypE 在还原条件下更好地结合。总之，这些结果为电子库存适合驱动多电子氧化还原反应提供了全面的证据，这些反应是在支架复合物 HypCD 上组装 CN- 和 CO 配体所需的。