Biosynthesis of the [FeFe] hydrogenase active site (the 'H-cluster') requires the interplay of multiple proteins and small molecules. Among them, the radical S-adenosylmethionine enzyme HydG, a tyrosine lyase, has been proposed to generate a complex that contains an Fe(CO)₂(CN) moiety that is eventually incorporated into the H-cluster. Here we describe the characterization of an intermediate in the HydG reaction: a [4Fe–4S][(Cys)Fe(CO)(CN)] species, 'Complex A', in which a CO, a CN⁻ and a cysteine (Cys) molecule bind to the unique 'dangler' Fe site of the auxiliary [5Fe–4S] cluster of HydG. The identification of this intermediate—the first organometallic precursor to the H-cluster—validates the previously hypothesized HydG reaction cycle and provides a basis for elucidating the biosynthetic origin of other moieties of the H-cluster.

[FeFe]氢化酶活性位点（“ H-簇”）的生物合成需要多种蛋白质和小分子的相互作用。其中，已经提出自由基S-腺苷甲硫氨酸酶HydG，酪氨酸裂解酶，以产生包含Fe（CO）₂（CN）部分的复合物，该复合物最终被掺入到H-簇中。在这里，我们描述一个中间的表征在HYDG反应：A [的4Fe-4S] [（CYS）的Fe（CO）（CN）]物种， '络合物A'，其中CO，CN的^-半胱氨酸（Cys）分子与HydG辅助[5Fe-4S]簇的唯一“悬挂物” Fe位点结合。该中间体（H簇的第一个有机金属前体）的鉴定验证了先前假设的HydG反应周期，并为阐明H簇其他部分的生物合成来源提供了基础。