The Catalytic Mechanism of Electron Bifurcating Electron Transfer Flavoproteins (ETFs) Involves an Intermediary Complex with NAD+

Paul King, David Mulder, Carolyn Lubner, Gerrit Schut, Nishya Mohammed-Raseek, Monika Tokmina-Lukaszewska, Diep Nguyen, Gina Lipscomb, John Hoben, Angela Patterson, John Peters, Brian Bothner, Anne-Frances Miller, Michael Adams

Research output: Contribution to journalArticlepeer-review

31 Scopus Citations


Electron bifurcation plays a key role in anaerobic energy metabolism, but it is a relatively new discovery, and only limited mechanistic information is available on the diverse enzymes that employ it. Herein, we focused on the bifurcating electron transfer flavoprotein (ETF) from the hyperthermophilic archaeon Pyrobaculum aerophilum. The EtfABCX enzyme complex couples NADH oxidation to the endergonic reduction of ferredoxin and exergonic reduction of menaquinone. We developed a model for the enzyme structure by using nondenaturing MS, cross-linking, and homology modeling in which EtfA, -B, and -C each contained FAD, whereas EtfX contained two [4Fe-4S] clusters. On the basis of analyses using transient absorption, EPR, and optical titrations with NADH or inorganic reductants with and without NAD, we propose a catalytic cycle involving formation of an intermediary NAD-bound complex. A charge transfer signal revealed an intriguing interplay of flavin semiquinones and a protein conformational change that gated electron transfer between the low- and high-potential pathways. We found that despite a common bifurcating flavin site, the proposed EtfABCX catalytic cycle is distinct from that of the genetically unrelated bifurcating NADH-dependent ferredoxin NADP oxidoreductase (NfnI). The two enzymes particularly differed in the role of NAD, the resting and bifurcating-ready states of the enzymes, how electron flow is gated, and the two two-electron cycles constituting the overall four-electron reaction. We conclude that P. aerophilum EtfABCX provides a model catalytic mechanism that builds on and extends previous studies of related bifurcating ETFs and can be applied to the large bifurcating ETF family.

Original languageAmerican English
Pages (from-to)3271-3283
Number of pages13
JournalJournal of Biological Chemistry
Issue number9
StatePublished - 1 Mar 2019

Bibliographical note

Publisher Copyright:
© 2019 American Society for Biochemistry and Molecular Biology Inc. All Rights Reserved.

NREL Publication Number

  • NREL/JA-2700-72604


  • bifurcation
  • bioenergetics
  • electron paramagnetic resonance (EPR)
  • electron transport
  • extreme thermophile
  • flavin
  • flavoprotein archaea
  • radical


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