Two distinct ferredoxins are essential for nitrogen fixation by the iron nitrogenase in .

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 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, strains with deletions of ferredoxin () and flavodoxin () genes were constructed to investigate their roles in nitrogen fixation by the Fe-nitrogenase. deletion strains were characterized by monitoring diazotrophic growth and Fe-nitrogenase activity . Only deletions of or resulted in slower growth and reduced Fe-nitrogenase activity, whereas the double deletion of both and abolished diazotrophic growth. Differences in the proteomes of ∆ and ∆ strains, in conjunction with differing plasmid complementation behaviors of and 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 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 , 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.