Electron Transfer to Nitrogenase in Different Genomic and Metabolic Backgrounds

Nitrogenase catalyzes the reduction of dinitrogen (N₂) using low-potential electrons from ferredoxin (Fd) or flavodoxin (Fld) through an ATP-dependent process. Since its emergence in an anaerobic chemoautotroph, this oxygen (O₂)-sensitive enzyme complex has evolved to operate in a variety of genomic and metabolic backgrounds, including those of aerobes, anaerobes, chemotrophs, and phototrophs. However, whether pathways of electron delivery to nitrogenase are influenced by these different metabolic backgrounds is not well understood. Here, we report the distribution of homologs of Fds, Flds, and Fd-/Fld-reducing enzymes in 359 genomes of putative N₂ fixers (diazotrophs). Six distinct lineages of nitrogenase were identified, and their distributions largely corresponded to differences in the host cells' ability to integrate O₂ or light into energy metabolism. The predicted pathways of electron transfer to nitrogenase in aerobes, facultative anaerobes, and phototrophs varied from those in anaerobes at the levels of Fds/Flds used to reduce nitrogenase, the enzymes that generate reduced Fds/Flds, and the putative substrates of these enzymes. Proteins that putatively reduce Fd with hydrogen or pyruvate were enriched in anaerobes, while those that reduce Fd with NADH/NADPH were enriched in aerobes, facultative anaerobes, and anoxygenic phototrophs. The energy metabolism of aerobic, facultatively anaerobic, and anoxygenic phototrophic diazotrophs often yields reduced NADH/NADPH that is not sufficiently reduced to drive N₂ reduction. At least two mechanisms have been acquired by these taxa to overcome this limitation and to generate electrons with potentials capable of reducing Fd. These include the bifurcation of electrons or the coupling of Fd reduction to reverse ion translocation.IMPORTANCE Nitrogen fixation supplies fixed nitrogen to cells from a variety of genomic and metabolic backgrounds, including those of aerobes, facultative anaerobes, chemotrophs, and phototrophs. Here, using informatics approaches applied to genomic data, we show that pathways of electron transfer to nitrogenase in metabolically diverse diazotrophic taxa have diversified primarily in response to host cells' acquired ability to integrate O₂ or light into their energy metabolism. The acquisition of two key enzyme complexes enabled aerobic and facultatively anaerobic phototrophic taxa to generate electrons of sufficiently low potential to reduce nitrogenase: the bifurcation of electrons via the Fix complex or the coupling of Fd reduction to reverse ion translocation via the Rhodobacter nitrogen fixation (Rnf) complex.