skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

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

Abstract

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 non-denaturing 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,more » how electron flow is gated, and in the two two-electron cycles constituting the overall four-electron reaction. Here, we conclude that P. aerophilum EtfABCX provides a model catalytic mechanism that builds on and extends previous studies of related bifurcating ETF's and can be applied to the large bifurcating ETF family.« less

Authors:
 [1];  [2];  [3];  [4];  [1];  [1];  [2];  [3]; ORCiD logo [4]; ORCiD logo [4];  [5];  [3]; ORCiD logo [2]; ORCiD logo [1]
  1. Univ. of Georgia, Athens, GA (United States)
  2. Univ. of Kentucky, Lexington, KY (United States)
  3. Montana State Univ., Bozeman, MT (United States)
  4. National Renewable Energy Lab. (NREL), Golden, CO (United States)
  5. Washington State Univ., Pullman, WA (United States)
Publication Date:
Research Org.:
Energy Frontier Research Centers (EFRC) (United States). Center for Biological Electron Transfer and Catalysis (BETCy); National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES)
OSTI Identifier:
1490994
Report Number(s):
NREL/JA-2700-72604
Journal ID: ISSN 0021-9258
Grant/Contract Number:  
AC36-08GO28308
Resource Type:
Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 294; Journal Issue: 9; Journal ID: ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular Biology
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; bifurcation; electron paramagnetic resonance (EPR); electron transport; flavoprotein archaea; extreme thermophile; bioenergetics; flavin; radical

Citation Formats

Schut, Gerrit J., Mohamed-Raseek, Nishya, Tokmina-Lukaszewska, Monika, Mulder, David W., Nguyen, Diep M. N., Lipscomb, Gina L., Hoben, John P., Patterson, Angela, Lubner, Carolyn E., King, Paul W., Peters, John W., Bothner, Brian, Miller, Anne -Frances, and Adams, Michael W. W. The catalytic mechanism of electron bifurcating electron transfer flavoproteins (ETFs) involves an intermediary complex with NAD+. United States: N. p., 2018. Web. doi:10.1074/jbc.RA118.005653.
Schut, Gerrit J., Mohamed-Raseek, Nishya, Tokmina-Lukaszewska, Monika, Mulder, David W., Nguyen, Diep M. N., Lipscomb, Gina L., Hoben, John P., Patterson, Angela, Lubner, Carolyn E., King, Paul W., Peters, John W., Bothner, Brian, Miller, Anne -Frances, & Adams, Michael W. W. The catalytic mechanism of electron bifurcating electron transfer flavoproteins (ETFs) involves an intermediary complex with NAD+. United States. doi:10.1074/jbc.RA118.005653.
Schut, Gerrit J., Mohamed-Raseek, Nishya, Tokmina-Lukaszewska, Monika, Mulder, David W., Nguyen, Diep M. N., Lipscomb, Gina L., Hoben, John P., Patterson, Angela, Lubner, Carolyn E., King, Paul W., Peters, John W., Bothner, Brian, Miller, Anne -Frances, and Adams, Michael W. W. Wed . "The catalytic mechanism of electron bifurcating electron transfer flavoproteins (ETFs) involves an intermediary complex with NAD+". United States. doi:10.1074/jbc.RA118.005653. https://www.osti.gov/servlets/purl/1490994.
@article{osti_1490994,
title = {The catalytic mechanism of electron bifurcating electron transfer flavoproteins (ETFs) involves an intermediary complex with NAD+},
author = {Schut, Gerrit J. and Mohamed-Raseek, Nishya and Tokmina-Lukaszewska, Monika and Mulder, David W. and Nguyen, Diep M. N. and Lipscomb, Gina L. and Hoben, John P. and Patterson, Angela and Lubner, Carolyn E. and King, Paul W. and Peters, John W. and Bothner, Brian and Miller, Anne -Frances and Adams, Michael W. W.},
abstractNote = {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 non-denaturing 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 in the two two-electron cycles constituting the overall four-electron reaction. Here, we conclude that P. aerophilum EtfABCX provides a model catalytic mechanism that builds on and extends previous studies of related bifurcating ETF's and can be applied to the large bifurcating ETF family.},
doi = {10.1074/jbc.RA118.005653},
journal = {Journal of Biological Chemistry},
number = 9,
volume = 294,
place = {United States},
year = {2018},
month = {12}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 3 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Structure and Function of the Nuclear Pore Complex Cytoplasmic mRNA Export Platform
journal, November 2016


Interaction between NADH and electron-transferring flavoprotein from Megasphaera elsdenii
journal, March 2013

  • Sato, K.; Nishina, Y.; Shiga, K.
  • Journal of Biochemistry, Vol. 153, Issue 6
  • DOI: 10.1093/jb/mvt026

The electron transfer flavoprotein: Ubiquinone oxidoreductases
journal, December 2010

  • Watmough, Nicholas J.; Frerman, Frank E.
  • Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol. 1797, Issue 12
  • DOI: 10.1016/j.bbabio.2010.10.007

Flavin-Based Electron Bifurcation, A New Mechanism of Biological Energy Coupling
journal, February 2018


Mechanistic insights into energy conservation by flavin-based electron bifurcation
journal, April 2017

  • Lubner, Carolyn E.; Jennings, David P.; Mulder, David W.
  • Nature Chemical Biology, Vol. 13, Issue 6
  • DOI: 10.1038/nchembio.2348

Electron Bifurcation: Thermodynamics and Kinetics of Two-Electron Brokering in Biological Redox Chemistry
journal, August 2017


Classic Spotlight: Electron Bifurcation, a Unifying Concept for Energy Conservation in Anaerobes
journal, April 2016


Comparative cross-linking and mass spectrometry of an intact F-type ATPase suggest a role for phosphorylation
journal, June 2013

  • Schmidt, Carla; Zhou, Min; Marriott, Hazel
  • Nature Communications, Vol. 4, Issue 1
  • DOI: 10.1038/ncomms2985

Probing the solution structure of Factor H using hydroxyl radical protein footprinting and cross-linking
journal, June 2016

  • Baud, Anna; Gonnet, Florence; Salard, Isabelle
  • Biochemical Journal, Vol. 473, Issue 12
  • DOI: 10.1042/BCJ20160225

Respiratory quinones in A rchaea : phylogenetic distribution and application as biomarkers in the marine environment : Archaeal quinones in cultures and environment
journal, December 2015

  • Elling, Felix J.; Becker, Kevin W.; Könneke, Martin
  • Environmental Microbiology, Vol. 18, Issue 2
  • DOI: 10.1111/1462-2920.13086

Natural engineering principles of electron tunnelling in biological oxidation–reduction
journal, November 1999

  • Page, Christopher C.; Moser, Christopher C.; Chen, Xiaoxi
  • Nature, Vol. 402, Issue 6757
  • DOI: 10.1038/46972

Use of protein cross-linking and radiolytic footprinting to elucidate PsbP and PsbQ interactions within higher plant Photosystem II
journal, October 2014

  • Mummadisetti, Manjula P.; Frankel, Laurie K.; Bellamy, Henry D.
  • Proceedings of the National Academy of Sciences, Vol. 111, Issue 45
  • DOI: 10.1073/pnas.1415165111

H/D exchange mass spectrometry and statistical coupling analysis reveal a role for allostery in a ferredoxin-dependent bifurcating transhydrogenase catalytic cycle
journal, January 2018

  • Berry, Luke; Poudel, Saroj; Tokmina-Lukaszewska, Monika
  • Biochimica et Biophysica Acta (BBA) - General Subjects, Vol. 1862, Issue 1
  • DOI: 10.1016/j.bbagen.2017.10.002

The Phyre2 web portal for protein modeling, prediction and analysis
journal, May 2015

  • Kelley, Lawrence A.; Mezulis, Stefans; Yates, Christopher M.
  • Nature Protocols, Vol. 10, Issue 6
  • DOI: 10.1038/nprot.2015.053

Evidence That the P i Release Event Is the Rate-Limiting Step in the Nitrogenase Catalytic Cycle
journal, June 2016


Reduction of ferredoxin or oxygen by flavin-based electron bifurcation in Megasphaera elsdenii
journal, May 2015

  • Chowdhury, Nilanjan P.; Kahnt, Jörg; Buckel, Wolfgang
  • The FEBS Journal, Vol. 282, Issue 16
  • DOI: 10.1111/febs.13308

Natural Competence in the Hyperthermophilic Archaeon Pyrococcus furiosus Facilitates Genetic Manipulation: Construction of Markerless Deletions of Genes Encoding the Two Cytoplasmic Hydrogenases
journal, February 2011

  • Lipscomb, Gina L.; Stirrett, Karen; Schut, Gerrit J.
  • Applied and Environmental Microbiology, Vol. 77, Issue 7, p. 2232-2238
  • DOI: 10.1128/AEM.02624-10

Suppression of Electron Transfer to Dioxygen by Charge Transfer and Electron Transfer Complexes in the FAD-dependent Reductase Component of Toluene Dioxygenase
journal, September 2012

  • Lin, Tzong-Yuan; Werther, Tobias; Jeoung, Jae-Hun
  • Journal of Biological Chemistry, Vol. 287, Issue 45
  • DOI: 10.1074/jbc.M112.374918

The CRISPR RNA-guided surveillance complex in Escherichia coli accommodates extended RNA spacers
journal, May 2016

  • Luo, Michelle L.; Jackson, Ryan N.; Denny, Steven R.
  • Nucleic Acids Research
  • DOI: 10.1093/nar/gkw421

Coupled Ferredoxin and Crotonyl Coenzyme A (CoA) Reduction with NADH Catalyzed by the Butyryl-CoA Dehydrogenase/Etf Complex from Clostridium kluyveri
journal, November 2007

  • Li, F.; Hinderberger, J.; Seedorf, H.
  • Journal of Bacteriology, Vol. 190, Issue 3
  • DOI: 10.1128/JB.01417-07

Unification of [FeFe]-hydrogenases into three structural and functional groups
journal, September 2016

  • Poudel, Saroj; Tokmina-Lukaszewska, Monika; Colman, Daniel R.
  • Biochimica et Biophysica Acta (BBA) - General Subjects, Vol. 1860, Issue 9
  • DOI: 10.1016/j.bbagen.2016.05.034

Molecular basis of the flavin-based electron-bifurcating caffeyl-CoA reductase reaction
journal, February 2018

  • Demmer, Julius K.; Bertsch, Johannes; Öppinger, Christian
  • FEBS Letters, Vol. 592, Issue 3
  • DOI: 10.1002/1873-3468.12971

Defining Electron Bifurcation in the Electron-Transferring Flavoprotein Family
journal, August 2017

  • Garcia Costas, Amaya M.; Poudel, Saroj; Miller, Anne-Frances
  • Journal of Bacteriology, Vol. 199, Issue 21
  • DOI: 10.1128/JB.00440-17

UCSF Chimera?A visualization system for exploratory research and analysis
journal, January 2004

  • Pettersen, Eric F.; Goddard, Thomas D.; Huang, Conrad C.
  • Journal of Computational Chemistry, Vol. 25, Issue 13
  • DOI: 10.1002/jcc.20084

Role of specific residues in coenzyme binding, charge–transfer complex formation, and catalysis in Anabaena ferredoxin NADP+-reductase
journal, September 2010

  • Peregrina, José Ramón; Sánchez-Azqueta, Ana; Herguedas, Beatriz
  • Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol. 1797, Issue 9
  • DOI: 10.1016/j.bbabio.2010.05.006

Purification and characterization of the tungsten enzyme aldehyde:ferredoxin oxidoreductase from the hyperthermophilic denitrifier Pyrobaculum aerophilum
journal, March 2005

  • Hagedoorn, Peter L.; Chen, Tianhong; Schröder, Imke
  • JBIC Journal of Biological Inorganic Chemistry, Vol. 10, Issue 3
  • DOI: 10.1007/s00775-005-0637-5

A simple method for the determination of reduction potentials in heme proteins
journal, January 2014


Energy Conservation via Electron-Transferring Flavoprotein in Anaerobic Bacteria
journal, November 2007

  • Herrmann, G.; Jayamani, E.; Mai, G.
  • Journal of Bacteriology, Vol. 190, Issue 3
  • DOI: 10.1128/JB.01422-07

Phylogenetic characterization of the ubiquitous electron transfer flavoprotein families ETF-α and ETF-β
journal, June 1995


The Electron Bifurcating FixABCX Protein Complex from Azotobacter vinelandii : Generation of Low-Potential Reducing Equivalents for Nitrogenase Catalysis
journal, August 2017


Distinct properties underlie flavin-based electron bifurcation in a novel electron transfer flavoprotein FixAB from Rhodopseudomonas palustris
journal, February 2018

  • Duan, H. Diessel; Lubner, Carolyn E.; Tokmina-Lukaszewska, Monika
  • Journal of Biological Chemistry, Vol. 293, Issue 13
  • DOI: 10.1074/jbc.RA117.000707

From low- to high-potential bioenergetic chains: Thermodynamic constraints of Q-cycle function
journal, September 2016

  • Bergdoll, Lucie; ten Brink, Felix; Nitschke, Wolfgang
  • Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol. 1857, Issue 9
  • DOI: 10.1016/j.bbabio.2016.06.006

An Electron-bifurcating Caffeyl-CoA Reductase
journal, March 2013

  • Bertsch, Johannes; Parthasarathy, Anutthaman; Buckel, Wolfgang
  • Journal of Biological Chemistry, Vol. 288, Issue 16
  • DOI: 10.1074/jbc.M112.444919

Heat Shock Protein 90 kDa (Hsp90) Has a Second Functional Interaction Site with the Mitochondrial Import Receptor Tom70
journal, July 2016

  • Zanphorlin, Leticia M.; Lima, Tatiani B.; Wong, Michael J.
  • Journal of Biological Chemistry, Vol. 291, Issue 36
  • DOI: 10.1074/jbc.M115.710137

Energy conservation via electron bifurcating ferredoxin reduction and proton/Na+ translocating ferredoxin oxidation
journal, February 2013

  • Buckel, Wolfgang; Thauer, Rudolf K.
  • Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol. 1827, Issue 2
  • DOI: 10.1016/j.bbabio.2012.07.002

Studies on the Mechanism of Electron Bifurcation Catalyzed by Electron Transferring Flavoprotein (Etf) and Butyryl-CoA Dehydrogenase (Bcd) of Acidaminococcus fermentans
journal, December 2013

  • Chowdhury, Nilanjan Pal; Mowafy, Amr M.; Demmer, Julius K.
  • Journal of Biological Chemistry, Vol. 289, Issue 8
  • DOI: 10.1074/jbc.M113.521013

A novel mode of lactate metabolism in strictly anaerobic bacteria: A novel mode of lactate metabolism in anaerobes
journal, May 2014

  • Weghoff, Marie Charlotte; Bertsch, Johannes; Müller, Volker
  • Environmental Microbiology, Vol. 17, Issue 3
  • DOI: 10.1111/1462-2920.12493

On the nature of organic and inorganic centers that bifurcate electrons, coupling exergonic and endergonic oxidation–reduction reactions
journal, January 2018

  • Peters, John W.; Beratan, David N.; Schut, Gerrit J.
  • Chemical Communications, Vol. 54, Issue 33
  • DOI: 10.1039/C8CC01530A

The Cac1 subunit of histone chaperone CAF-1 organizes CAF-1-H3/H4 architecture and tetramerizes histones
journal, September 2016


Mass spectrometry-based carboxyl footprinting of proteins: Method evaluation
journal, February 2012

  • Zhang, Hao; Wen, Jianzhong; Huang, Richard Y-C.
  • International Journal of Mass Spectrometry, Vol. 312
  • DOI: 10.1016/j.ijms.2011.07.015

Possible molecular mechanisms of the protonmotive function of cytochrome systems
journal, October 1976


A new era for electron bifurcation
journal, December 2018


SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information
journal, April 2014

  • Biasini, Marco; Bienert, Stefan; Waterhouse, Andrew
  • Nucleic Acids Research, Vol. 42, Issue W1
  • DOI: 10.1093/nar/gku340

Genome sequence of the hyperthermophilic crenarchaeon Pyrobaculum aerophilum
journal, January 2002

  • Fitz-Gibbon, S. T.; Ladner, H.; Kim, U. -J.
  • Proceedings of the National Academy of Sciences, Vol. 99, Issue 2
  • DOI: 10.1073/pnas.241636498

The semiquinone swing in the bifurcating electron transferring flavoprotein/butyryl-CoA dehydrogenase complex from Clostridium difficile
journal, November 2017

  • Demmer, Julius K.; Pal Chowdhury, Nilanjan; Selmer, Thorsten
  • Nature Communications, Vol. 8, Issue 1
  • DOI: 10.1038/s41467-017-01746-3

Two functionally distinct NADP + -dependent ferredoxin oxidoreductases maintain the primary redox balance of Pyrococcus furiosus
journal, July 2017

  • Nguyen, Diep M. N.; Schut, Gerrit J.; Zadvornyy, Oleg A.
  • Journal of Biological Chemistry, Vol. 292, Issue 35
  • DOI: 10.1074/jbc.M117.794172

Electron bifurcation
journal, April 2016

  • Peters, John W.; Miller, Anne-Frances; Jones, Anne K.
  • Current Opinion in Chemical Biology, Vol. 31
  • DOI: 10.1016/j.cbpa.2016.03.007

Regulation of the mechanism of Type-II NADH: Quinone oxidoreductase from S. aureus
journal, June 2018


D-Lactate dehydrogenase of Peptostreptococcus elsdenii.
journal, January 1975


Pyrobaculum aerophilum sp. nov., a novel nitrate-reducing hyperthermophilic archaeum.
journal, January 1993


    Works referencing / citing this record:

    The oxygen reduction reaction catalyzed by Synechocystis sp. PCC 6803 flavodiiron proteins
    journal, January 2019

    • Brown, Katherine A.; Guo, Zhanjun; Tokmina-Lukaszewska, Monika
    • Sustainable Energy & Fuels, Vol. 3, Issue 11
    • DOI: 10.1039/c9se00523d

    Spectroscopic, thermodynamic and computational evidence of the locations of the FADs in the nitrogen fixation-associated electron transfer flavoprotein
    journal, January 2019

    • Mohamed-Raseek, Nishya; Duan, H. Diessel; Hildebrandt, Peter
    • Chemical Science, Vol. 10, Issue 33
    • DOI: 10.1039/c9sc00942f

    Spectroscopic, thermodynamic and computational evidence of the locations of the FADs in the nitrogen fixation-associated electron transfer flavoprotein
    journal, January 2019

    • Mohamed-Raseek, Nishya; Duan, H. Diessel; Hildebrandt, Peter
    • Chemical Science, Vol. 10, Issue 33
    • DOI: 10.1039/c9sc00942f

    Electron bifurcation: progress and grand challenges
    journal, January 2019

    • Yuly, Jonathon L.; Lubner, Carolyn E.; Zhang, Peng
    • Chemical Communications, Vol. 55, Issue 79
    • DOI: 10.1039/c9cc05611d