Electron transfer to nitrogenase in Paenibacillus polymyxa

Nitrogenase requires low-potential electrons to reduce N₂ to produce ammonia. The direct electron donors to nitrogenase are reduced ferredoxin (Fd) and flavodoxin (Fld). The enzymes that reduce Fd or Fld for N₂ fixation mainly include pyruvate-flavodoxin (ferredoxin) oxidoreductase (PFOR), ferredoxin-NADP⁺ reductase (FNR), Rhodobacter nitrogen fixation protein complex (Rnf), and bifurcating electron transfer flavoprotein complex (Fix). Paenibacillus polymyxa is a model for studies of nitrogen fixation, but the pathways of electron transport to nitrogenase are unknown. In this study, we characterized the electron transport pathway to nitrogenase in P. polymyxa. Deletion analysis showed that among 15 genes encoding Fds, Fld, and Fd-/Fld-reducing enzymes, only cbiW gene and pfoAB operon decreased nitrogenase activity (31% and 37% relative to wild type, respectively). Triple deletion of cbiW and pfoAB led to a reduction of nitrogenase activity by 61%. Elemental analysis and ultraviolet-visible absorption spectroscopy revealed that the purified Fd protein encoded by cbiW is a [2Fe-2S] Fd. The purified Fd protein was able to substitute for Klebsiella NifF (Fld) to transfer electrons from pyruvate to Klebsiella nitrogenase in vitro. The pfoAB operon encodes PFOR, which is composed of two subunits. Klebsiella oxytoca NifJ (PFOR) was able to support nitrogen fixation in the ΔpfoAB mutant. These findings suggest that [2Fe-2S] Fd encoded by cbiW and PFOR encoded by the pfoAB operon constitute an electron transport pathway to Paenibacillus nitrogenase.

Gram-positive and facultatively anaerobic Paenibacillus polymyxa is a model diazotroph whose nif (nitrogen fixation) genes encoding nitrogenase have been used for engineering yeast and plants. However, the pathway of electron transfer to Paenibacillus nitrogenase is unknown. In this study, we demonstrate that Fd encoded by cbiW and PFOR encoded by the pfoAB operon constitute an electron transport pathway to Paenibacillus nitrogenase. Fd encoded by cbiW is a [2Fe-2S] Fd, and PFOR encoded by the pfoAB operon is a heterodimer. In contrast, in many gram-negative diazotrophs, [4Fe-4S] Fds are widely used as electron donors to nitrogenase, and PFORs (NifJs) are homodimers. These results suggest that there may be some differences in the mechanisms of electron transfer to nitrogenase between gram-negative and gram-positive bacteria. The identified electron transport components in P. polymyxa will have great potential for use in engineering eukaryotes.