The M-cluster is the [(homocitrate)MoFe₇S₉C] active site of nitrogenase that is derived from an 8Fe core assembled viacoupling and rearrangement of two [Fe₄S₄] clusters concomitant with the insertion of an interstitial carbon and a ‘ninth sulfur’. Combining synthetic [Fe₄S₄] clusters with an assembly protein template, here we show that sulfite can give rise to the ninth sulfur that is incorporated in the catalytically important belt region of the cofactor after the radical S-adenosyl-l-methionine-dependent carbide insertion and the concurrent 8Fe-core rearrangement have already taken place. Based on the differential reactivity of the formed cluster species, we also propose a new [Fe₈S₈C] cluster intermediate, the L*-cluster, which is similar to the [Fe₈S₉C] L-cluster, but lacks the ninth sulfur from sulfite. This work provides a semi-synthetic tool for protein reconstitution that could be widely applicable for the functional analysis of other FeS systems.

M簇是[（单价）MoFe ₇ S ₉ C]的固氮酶活性位点，它是通过两个铁[Fe ₄ S ₄ ]团簇的偶联和重排组装而成的8Fe核的核心，同时插入间隙碳和碳原子。 “第九硫”。将合成的[Fe ₄ S ₄ ]簇与组装蛋白模板结合起来，在这里我们显示亚硫酸盐可以产生第九个硫，该硫在自由基S-腺苷基I之后被引入到辅因子的催化重要带区中甲硫氨酸依赖的硬质合金插入和同时发生的8Fe核重排已经发生。基于形成的簇物种的差异反应性，我们还提出了一种新的[Fe ₈ S ₈ C]簇中间体L *簇，与[Fe ₈ S ₉ C] L簇相似，但缺乏亚硫酸盐中的第九种硫。这项工作为蛋白质重建提供了一种半合成工具，可以广泛应用于其他FeS系统的功能分析。