The catalytic mechanism of electron-bifurcating electron transfer flavoproteins (ETFs) involves an intermediary complex with NAD

Gerrit J. Schut, Nishya Mohamed-Raseek, Monika Tokmina-Lukaszewska, David W. Mulder, Diep M.N. Nguyen, Gina L. Lipscomb, John P. Hoben, Angela Patterson, Carolyn E. Lubner, Paul W. King, John W. Peters, Brian Bothner, Anne Frances Miller, Michael W.W. Adams

Research output: Contribution to journalArticlepeer-review

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

Bibliographical note

Funding Information:
This work and all authors were supported as part of the Biological and Electron Transfer and Catalysis (BETCy) EFRC, an Energy Frontier Research Center funded by the United States Department of Energy, Office of Science, Basic Energy Sciences under Award DE-SC0012518. Additional support was pro-vided by the Research Challenge Trust fund of Kentucky (NM-R) and by the “Unifying Concepts in Catalysis” cluster of excellence (UNICAT) and the Tech-nische Universität-Berlin (to A.-F. M.). This work was authored in part by the National Renewable Energy Laboratory, operated by Alliance for Sustainable Energy, LLC, for the U.S. Department of Energy (DOE) under Contract No. DE-AC36–08GO28308 (to D. W. M., C. E. L., and P. W. K.). Funding for TAS and EPR measurements provided by the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences, and Biosciences (D. W. M., C. E. L., and P. W. K.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsi-bility of the authors and does not necessarily represent the official views of the National Institutes of Health. The views expressed in the article do not neces-sarily represent the views of the DOE or the U.S. Government.

Funding Information:
Acknowledgments—The Mass Spectrometry Facility at Montana State University is supported in part by the Murdock Charitable Trust and National Institutes of Health IDEA Program Grant P20GM103474. We thank the Microfabrication Facility of Montana State University for help in preparation of gold-coated borosilica capillaries for noncovalent MS and Zachary Wood (University of Georgia) for use of the upcycled Agilent HP 8453.

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

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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