The nitrogenase metalloenzyme family, essential for supplying fixed nitrogen to the biosphere, is one of life's key biogeochemical innovations. The three forms of nitrogenase differ in their metal dependence, each binding either a FeMo‐, FeV‐, or FeFe‐cofactor where the reduction of dinitrogen takes place. The history of nitrogenase metal dependence has been of particular interest due to the possible implication that ancient marine metal availabilities have significantly constrained nitrogenase evolution over geologic time. Here, we reconstructed the evolutionary history of nitrogenases, and combined phylogenetic reconstruction, ancestral sequence inference, and structural homology modeling to evaluate the potential metal dependence of ancient nitrogenases. We find that active‐site sequence features can reliably distinguish extant Mo‐nitrogenases from V‐ and Fe‐nitrogenases and that inferred ancestral sequences at the deepest nodes of the phylogeny suggest these ancient proteins most resemble modern Mo‐nitrogenases. Taxa representing early‐branching nitrogenase lineages lack one or more biosynthetic nifE and nifN genes that both contribute to the assembly of the FeMo‐cofactor in studied organisms, suggesting that early Mo‐nitrogenases may have utilized an alternate and/or simplified pathway for cofactor biosynthesis. Our results underscore the profound impacts that protein‐level innovations likely had on shaping global biogeochemical cycles throughout the Precambrian, in contrast to organism‐level innovations that characterize the Phanerozoic Eon.

中文翻译：

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重建固氮酶的进化史：祖先钼辅助因子利用的证据。

固氮酶金属酶家族对于向生物圈提供固定氮至关重要，是生命中关键的生物地球化学创新之一。三种形式的固氮酶对金属的依赖性不同，每种固氮因子都与FeMo-，FeV-或FeFe-辅因子结合，从而导致二氮的还原。由于可能暗示古代海洋金属的可用性已经大大限制了地质时间内硝化酶的演化，因此对硝化酶金属依赖性的历史一直特别感兴趣。在这里，我们重建了固氮酶的进化历史，并结合了系统发育重建，祖先序列推断和结构同源性建模，以评估古代固氮酶的潜在金属依赖性。我们发现活性位点序列特征可以可靠地将现存的Mo-氮酶与V-和Fe-氮酶区分开，并且在系统发育的最深节点推断出的祖先序列表明这些古老的蛋白质最类似于现代Mo-氮酶。代表早期分支固氮酶谱系的分类单元缺乏一种或多种生物合成nifE的和nifN基因既有助于装配中的铁钼辅因子的研究中的生物体，表明早期的Mo-固氮可能利用的替换和/或简化途径辅因子的生物合成。我们的结果强调了蛋白质水平的创新可能对塑造整个前寒武纪的全球生物地球化学循环产生深远的影响，这与生代古生化时期的生物体水平的创新形成了鲜明的对比。