Cryoelectron microscopy structure and mechanism of the membrane-associated electron-bifurcating flavoprotein Fix/EtfABCX

Proc Natl Acad Sci U S A. 2021 Jan 12;118(2):e2016978118. doi: 10.1073/pnas.2016978118.


The electron-transferring flavoprotein-menaquinone oxidoreductase ABCX (EtfABCX), also known as FixABCX for its role in nitrogen-fixing organisms, is a member of a family of electron-transferring flavoproteins that catalyze electron bifurcation. EtfABCX enables endergonic reduction of ferredoxin (E°' ∼-450 mV) using NADH (E°' -320 mV) as the electron donor by coupling this reaction to the exergonic reduction of menaquinone (E°' -80 mV). Here we report the 2.9 Å structure of EtfABCX, a membrane-associated flavin-based electron bifurcation (FBEB) complex, from a thermophilic bacterium. EtfABCX forms a superdimer with two membrane-associated EtfCs at the dimer interface that contain two bound menaquinones. The structure reveals that, in contrast to previous predictions, the low-potential electrons bifurcated from EtfAB are most likely directly transferred to ferredoxin, while high-potential electrons reduce the quinone via two [4Fe-4S] clusters in EtfX. Surprisingly, EtfX shares remarkable structural similarity with mammalian [4Fe-4S] cluster-containing ETF ubiquinone oxidoreductase (ETF-QO), suggesting an unexpected evolutionary link between bifurcating and nonbifurcating systems. Based on this structure and spectroscopic studies of a closely related EtfABCX, we propose a detailed mechanism of the catalytic cycle and the accompanying structural changes in this membrane-associated FBEB system.

Keywords: Thermotoga; electron bifurcation; flavin; iron-sulfur cluster; menaquinone.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacterial Proteins / metabolism
  • Catalysis
  • Cryoelectron Microscopy / methods
  • Electron Transport
  • Electron-Transferring Flavoproteins / metabolism*
  • Electrons
  • Ferredoxins / metabolism
  • Flavins / metabolism
  • Flavoproteins / metabolism
  • Iron-Sulfur Proteins / metabolism
  • Models, Molecular
  • NAD / metabolism
  • Nitrogen Fixation / physiology
  • Oxidation-Reduction
  • Pyrococcus furiosus / metabolism
  • Quinone Reductases / metabolism*
  • Quinone Reductases / physiology
  • Quinone Reductases / ultrastructure*
  • Vitamin K 2 / metabolism


  • Bacterial Proteins
  • Electron-Transferring Flavoproteins
  • Ferredoxins
  • Flavins
  • Flavoproteins
  • Iron-Sulfur Proteins
  • NAD
  • Vitamin K 2
  • Quinone Reductases