Molecular innovations within key metabolisms can have profound impacts on element cycling and ecological distribution. Yet, much of the molecular foundations of early evolved enzymes and metabolisms are unknown. Here, we bring one such mystery to relief by probing the birth and evolution of the G-subunit protein, an integral component of certain members of the nitrogenase family, the only enzymes capable of biological nitrogen fixation. The G-subunit is a Paleoproterozoic-age orphan protein that appears more than 1 billion years after the origin of nitrogenases. We show that the G-subunit arose with novel nitrogenase metal dependence and the ecological expansion of nitrogen-fixing microbes following the transition in enviromental metal availabilities and atmospheric oxygenation that began ∼2.5 billion years ago. We identify molecular features that suggest early G-subunit proteins mediated cofactor or protein interactions required for novel metal dependency, priming ancient nitrogenases and their hosts to exploit these newly diversified geochemical environments. We further examined the degree of functional specialization in G-subunit evolution with extant and ancestral homologs using laboratory reconstruction experiments. Our results indicate that permanent recruitment of the orphan protein depended on the prior establishment of conserved molecular features and showcase how contingent evolutionary novelties might shape ecologically important microbial innovations.

中文翻译：

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大气氧化后孤儿固氮酶蛋白的出现

关键代谢中的分子创新可以对元素循环和生态分布产生深远的影响。然而，早期进化的酶和新陈代谢的许多分子基础尚不清楚。在这里，我们通过探索 G 亚基蛋白的诞生和进化来解开这样一个谜团，G 亚基蛋白是固氮酶家族某些成员的组成部分，而固氮酶家族是唯一能够进行生物固氮的酶。 G 亚基是古元古代的孤儿蛋白，在固氮酶起源 10 亿多年后出现。我们发现，随着约 25 亿年前开始的环境金属可用性和大气氧化的转变，G 亚基随着新型固氮酶金属依赖性和固氮微生物的生态扩张而出现。我们确定的分子特征表明早期 G 亚基蛋白介导新型金属依赖性所需的辅因子或蛋白质相互作用，从而启动古代固氮酶及其宿主利用这些新多样化的地球化学环境。我们使用实验室重建实验进一步检查了 G 亚基进化与现存和祖先同源物的功能专业化程度。我们的结果表明，孤儿蛋白的永久招募取决于先前建立的保守分子特征，并展示了偶然的进化新颖性如何塑造具有重要生态意义的微生物创新。