Two distinct ferredoxins are essential for nitrogen fixation by the iron nitrogenase inRhodobacter capsulatus

Abstract

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 characterised the electron transfer pathway to the Fe-nitrogenase inRhodobacter capsulatusvia 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. capsulatusstrains with deletions of ferredoxin (fdx) and flavodoxin (fld, nifF) genes were constructed to investigate their roles in nitrogen fixation by the Fe-nitrogenase.R. capsulatusdeletion strains were characterised by monitoring diazotrophic growth and Fe-nitrogenase activityin vivo. Only deletions offdxCorfdxNresulted in slower growth and reduced Fe-nitrogenase activity, whereas the double-deletion of bothfdxCandfdxNabolished diazotrophic growth. Differences in the proteomes of ΔfdxCand ΔfdxNstrains, in conjunction with differing plasmid complementation behaviours offdxCandfdxN, 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. Production of ammonia by diazotrophic organisms, harbouring 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, though we lack knowledge as to which proteins shuttle the electrons to nitrogenases within cells. Here, we characterised the electron transport to the iron (Fe)-nitrogenase in the model diazotrophRhodobacter capsulatus, showing that two distinct ferredoxins are very important for nitrogen fixation despite having different redox centres. Additionally, 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 behaviours.