固氮酶是唯一能够将气态氮固定为生物可利用的氨的酶,因此对于维持生命至关重要。固氮酶的催化作用需要大量的ATP和通过强烈还原铁氧还蛋白或黄素蛋白而提供的电子。我们对电子转移到固氮酶的机制的了解有限:几乎没有研究电子转移到铁(Fe)-固氮酶的机制。这里,我们通过蛋白质组分析表征了胶囊中铁固氮酶的电子转移途径,基因缺失,补充研究,和系统发育学。蛋白质组分析显示,在钼固氮酶敲除菌株中,在固氮条件下,四种铁氧还蛋白的上调依赖于铁固氮酶,与非固氮条件相比。基于这些发现,含铁氧还蛋白(fdx)和黄素氧还蛋白(fld,构建nifF)基因以研究其在Fe-固氮酶固氮中的作用。通过监测体内的重氮生长和Fe-固氮酶活性来表征荚膜R.只有fdxC或fdxN的缺失导致生长缓慢和铁固氮酶活性降低,而fdxC和fdxN的双重缺失消除了重氮生长。ΔfdxC和ΔfdxN菌株的蛋白质组差异,结合fdxC和fdxN的不同质粒互补行为,这表明这两个联邦调查局可能拥有不同的角色和职能。这些发现将指导未来的固氮酶电子传递系统的工程,目的是增加电子通量和产物形成。importancenitrogenases是必不可少的生物固氮,将大气中的氮气转化为生物可利用的氨。重氮生物产生氨,藏有固氮酶,对维持植物生长至关重要。因此,通过固氮酶了解固氮的细胞机制有很大的科学兴趣。硝化酶依靠高度还原的电子来催化,尽管我们缺乏关于哪些蛋白质将电子穿梭到细胞内的固氮酶的知识。这里,我们表征了模型重氮菌中电子向铁(Fe)-固氮酶的传输,表明尽管具有不同的氧化还原中心,但两种不同的铁氧还蛋白对于固氮非常重要。此外,我们的研究扩展了关于铁氧还蛋白是否具有功能冗余或在细胞内发挥不同作用的辩论。这里,我们观察到,基于缺失菌株的差异蛋白质组移位和不同的互补行为,两种必需铁皮苷可能具有不同的作用。
Nitrogenases are the only enzymes able to fix gaseous nitrogen into bioavailable ammonia and hence are essential for sustaining life. Catalysis by nitrogenases requires both a large amount of ATP and electrons donated by strongly reducing ferredoxins or flavodoxins. Our knowledge about the mechanisms of electron transfer to nitrogenase enzymes is limited: The electron transport to the iron (Fe)-nitrogenase has hardly been investigated. Here, we characterized the electron transfer pathway to the Fe-nitrogenase in Rhodobacter capsulatus via proteome analyses, genetic deletions, complementation studies, and phylogenetics. Proteome analyses revealed an upregulation of four ferredoxins under nitrogen-fixing conditions reliant on the Fe-nitrogenase in a molybdenum nitrogenase knockout strain, compared to non-nitrogen-fixing conditions. Based on these findings, R. capsulatus strains with deletions of ferredoxin (fdx) and flavodoxin (fld, nifF) genes were constructed to investigate their roles in nitrogen fixation by the Fe-nitrogenase. R. capsulatus deletion strains were characterized by monitoring diazotrophic growth and Fe-nitrogenase activity in vivo. Only deletions of fdxC or fdxN resulted in slower growth and reduced Fe-nitrogenase activity, whereas the double deletion of both fdxC and fdxN abolished diazotrophic growth. Differences in the proteomes of ∆fdxC and ∆fdxN strains, in conjunction with differing plasmid complementation behaviors of fdxC and fdxN, indicate that the two Fds likely possess different roles and functions. These findings will guide future engineering of the electron transport systems to nitrogenase enzymes, with the aim of increased electron flux and product formation.IMPORTANCENitrogenases are essential for biological nitrogen fixation, converting atmospheric nitrogen gas to bioavailable ammonia. The production of ammonia by diazotrophic organisms, harboring nitrogenases, is essential for sustaining plant growth. Hence, there is a large scientific interest in understanding the cellular mechanisms for nitrogen fixation via nitrogenases. Nitrogenases rely on highly reduced electrons to power catalysis, although we lack knowledge as to which proteins shuttle the electrons to nitrogenases within cells. Here, we characterized the electron transport to the iron (Fe)-nitrogenase in the model diazotroph Rhodobacter capsulatus, showing that two distinct ferredoxins are very important for nitrogen fixation despite having different redox centers. In addition, our research expands upon the debate on whether ferredoxins have functional redundancy or perform distinct roles within cells. Here, we observe that both essential ferredoxins likely have distinct roles based on differential proteome shifts of deletion strains and different complementation behaviors.