The Electron Bifurcating FixABCX Protein Complex from Azotobacter vinelandii: Generation of Low-Potential Reducing Equivalents for Nitrogenase Catalysis

Paul King, Carolyn Lubner, David Mulder, Rhesa Ledbetter, Amaya Garcia Costas, Monika Tokmina-Lukaszewska, Jacob Artz, Angela Patterson, Timothy Magnuson, Zackary Jay, H. Duan, Jacquelyn Miller, Mary Plunkett, John Hoben, Brett Barney, Ross Carlson, Anne-Frances Miller, Brian Bothner, John Peters, Lance Seefeldt

Research output: Contribution to journalArticlepeer-review

106 Scopus Citations


The biological reduction of dinitrogen (N2) to ammonia (NH3) by nitrogenase is an energetically demanding reaction that requires low-potential electrons and ATP; however, pathways used to deliver the electrons from central metabolism to the reductants of nitrogenase, ferredoxin or flavodoxin, remain unknown for many diazotrophic microbes. The FixABCX protein complex has been proposed to reduce flavodoxin or ferredoxin using NADH as the electron donor in a process known as electron bifurcation. Herein, the FixABCX complex from Azotobacter vinelandii was purified and demonstrated to catalyze an electron bifurcation reaction: oxidation of NADH (Em = -320 mV) coupled to reduction of flavodoxin semiquinone (Em = -460 mV) and reduction of coenzyme Q (Em = 10 mV). Knocking out fix genes rendered Δrnf A. vinelandii cells unable to fix dinitrogen, confirming that the FixABCX system provides another route for delivery of electrons to nitrogenase. Characterization of the purified FixABCX complex revealed the presence of flavin and iron-sulfur cofactors confirmed by native mass spectrometry, electron paramagnetic resonance spectroscopy, and transient absorption spectroscopy. Transient absorption spectroscopy further established the presence of a short-lived flavin semiquinone radical, suggesting that a thermodynamically unstable flavin semiquinone may participate as an intermediate in the transfer of an electron to flavodoxin. A structural model of FixABCX, generated using chemical cross-linking in conjunction with homology modeling, revealed plausible electron transfer pathways to both high- and low-potential acceptors. Overall, this study informs a mechanism for electron bifurcation, offering insight into a unique method for delivery of low-potential electrons required for energy-intensive biochemical conversions.

Original languageAmerican English
Pages (from-to)4177-4190
Number of pages14
Issue number32
StatePublished - 15 Aug 2017

Bibliographical note

Publisher Copyright:
© 2017 American Chemical Society.

NREL Publication Number

  • NREL/JA-2700-68237


  • electron paramagnetic resonance
  • electron transfer
  • EPR
  • flavoprotein
  • nitrogen fixation
  • oxidation-reduction
  • redox


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