Distinct Properties Underlie Flavin-Based Electron Bifurcation in a Novel Electron Transfer Flavoprotein FixAB from Rhodopseudomonas palustris

H. Diessel Duan, Carolyn E. Lubner, Monika Tokmina-Lukaszewska, George H. Gauss, Brian Bothner, Paul W. King, John W. Peters, Anne Frances Miller

Research output: Contribution to journalArticlepeer-review

20 Scopus Citations


A newly recognized third fundamental mechanism of energy conservation in biology, electron bifurcation, uses free energy from exergonic redox reactions to drive endergonic redox reactions. Flavin-based electron bifurcation furnishes low-potential electrons to demanding chemical reactions, such as reduction of dinitrogen to ammonia. We employed the heterodimeric fla-voenzyme FixAB from the diazotrophic bacterium Rhodopseudomonas palustris to elucidate unique properties that underpin flavin-based electron bifurcation. FixAB is distinguished from canonical electron transfer flavoproteins (ETFs) by a second FAD that replaces the AMP of canonical ETF. We exploited near-UV-visible CD spectroscopy to resolve signals from the different flavin sites in FixAB and to interrogate the putative bifurcating FAD. CD aided in assigning the measured reduction midpoint potentials (E° values) to individual flavins, and the E° values tested the accepted model regarding the redox properties required for bifurcation. We found that the higher-E° flavin displays sequential one-electron (1-e) reductions to anionic semiquinone and then to hydroquinone, consistent with the reactivity seen in canonical ETFs. In contrast, the lower-E° flavin displayed a single two-electron (2-e) reduction without detectable accumulation of semiquinone, consistent with unstable semiquinone states, as required for bifurcation. This is the first demonstration that a FixAB protein possesses the thermodynamic prerequisites for bifurcating activity, and the separation of distinct optical signatures for the two flavins lays a foundation for mechanistic studies to learn how electron flow can be directed in a protein environment. We propose that a novel optical signal observed at long wavelength may reflect electron delocalization between the two flavins.

Original languageAmerican English
Pages (from-to)4688-4701
Number of pages14
JournalJournal of Biological Chemistry
Issue number13
StatePublished - 2018

Bibliographical note

Publisher Copyright:
© 2018 American Society for Biochemistry and Molecular Biology Inc. All Rights Reserved.

NREL Publication Number

  • NREL/JA-2700-70488


  • circular dichroism
  • electron bifurcation
  • electron transfer
  • electron transfer flavorprotein
  • ETF
  • FixAB
  • flavin
  • flavorprotein
  • nitrogenase
  • reduction midpoint potential


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