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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.

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AbstractElectron bifurcation was first used to describe the electron-transfer events associated with the quinone-cycle (Q-cycle), which occurs in complex III of the electron transport chain. Decades later, flavin-based electron bifurcation (FBEB) was discovered, where flavin cofactors act in analogy to Q to drive the endergonic reduction of ferredoxin (Fd) or flavodoxin (Fld) by NAD(P)H. The soil microbeAzotobacter vinelandii is known to generate low-potential electrons in the form of reduced Fd or Fld using FBEB via the enzyme complex FixABCX. FixABCX in A. vinelandii (AvFixABCX) couples the exergonic reduction of coenzyme Q (E = +10 mV) to the endergonic reduction of Fd/Fld (E = ∼−450 mV) during NADH oxidation (E = −320 mV). In this work, we successfully optimized the expression of AvFixABCX in E. coli. We have employed protein thin-film electrochemistry, UV–vis spectrophotometry, electron paramagnetic resonance (EPR) spectroscopy, and transient absorption (TA) spectroscopy to elucidate the complete energy landscape, and structural characterization methods, cryo-electron microscopy (cryo-EM), small-angle X-ray scattering (SAXS), and cross-linking mass spectrometry (XL-MS) to show the dynamics of the complex energy landscape during AvFixABCX electron-transfer catalysis. Our results show that AvFixABCX contains two [4Fe-4S] clusters with disparate reduction potentials: the higher-potential cluster participates in the exergonic electron-transfer pathway, whereas the lower-potential cluster is off-pathway. We experimentally determined that AvFixABCX undergoes a conformationally gated, reversible FBEB mechanism to simultaneously reduce coenzyme Q and the semiquinone state of Fld during NADH oxidation.