Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Mechanistic insights into energy conservation by flavin-based electron bifurcation

Nature Chemical Biology volume 13, pages 655–659 (2017)Cite this article

Subjects

Abstract

The recently realized biochemical phenomenon of energy conservation through electron bifurcation provides biology with an elegant means to maximize utilization of metabolic energy. The mechanism of coordinated coupling of exergonic and endergonic oxidation–reduction reactions by a single enzyme complex has been elucidated through optical and paramagnetic spectroscopic studies revealing unprecedented features. Pairs of electrons are bifurcated over more than 1 volt of electrochemical potential by generating a low-potential, highly energetic, unstable flavin semiquinone and directing electron flow to an iron–sulfur cluster with a highly negative potential to overcome the barrier of the endergonic half reaction. The unprecedented range of thermodynamic driving force that is generated by flavin-based electron bifurcation accounts for unique chemical reactions that are catalyzed by these enzymes.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Pf NfnI structure and orientation of cofactors.
Figure 2: Exergonic branch for electron transfer between NADP(H) and NAD(H) in Pf NfnI.
Figure 3: Endergonic electron transfer between NADP(H) and Fd in Pf NfnI.
Figure 4: NADP(H) binding affects both short- and long-range interactions in Pf NfnI.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Buckel, W. & Thauer, R.K. Energy conservation via electron bifurcating ferredoxin reduction and proton/Na(+) translocating ferredoxin oxidation. Biochim. Biophys. Acta 1827, 94–113 (2013).

    Article  CAS  Google Scholar 

  2. Mitchell, P. The protonmotive Q cycle: a general formulation. FEBS Lett. 59, 137–139 (1975).

    Article  CAS  Google Scholar 

  3. Herrmann, G., Jayamani, E., Mai, G. & Buckel, W. Energy conservation via electron-transferring flavoprotein in anaerobic bacteria. J. Bacteriol. 190, 784–791 (2008).

    Article  CAS  Google Scholar 

  4. Peters, J.W., Miller, A.-F., Jones, A.K., King, P.W. & Adams, M.W. Electron bifurcation. Curr. Opin. Chem. Biol. 31, 146–152 (2016).

    Article  CAS  Google Scholar 

  5. Brandt, U. Bifurcated ubihydroquinone oxidation in the cytochrome bc 1 complex by proton-gated charge transfer. FEBS Lett. 387, 1–6 (1996).

    Article  CAS  Google Scholar 

  6. Mulkidjanian, A.Y. Activated Q-cycle as a common mechanism for cytochrome bc 1 and cytochrome b 6 f complexes. Biochim. Biophys. Acta 1797, 1858–1868 (2010).

    Article  CAS  Google Scholar 

  7. Demmer, J.K. et al. Insights into flavin-based electron bifurcation via the NADH-dependent reduced ferredoxin:NADP oxidoreductase structure. J. Biol. Chem. 290, 21985–21995 (2015).

    Article  CAS  Google Scholar 

  8. Wang, S., Huang, H., Moll, J. & Thauer, R.K. NADP+ reduction with reduced ferredoxin and NADP+ reduction with NADH are coupled via an electron-bifurcating enzyme complex in Clostridium kluyveri. J. Bacteriol. 192, 5115–5123 (2010).

    Article  CAS  Google Scholar 

  9. Chowdhury, N.P., Klomann, K., Seubert, A. & Buckel, W. Reduction of flavodoxin by electron bifurcation and sodium ion-dependent reoxidation by NAD+ catalyzed by ferredoxin-NAD+ reductase (Rnf). J. Biol. Chem. 291, 11993–12002 (2016).

    Article  CAS  Google Scholar 

  10. Chowdhury, N.P. et al. Studies on the mechanism of electron bifurcation catalyzed by electron transferring flavoprotein (Etf) and butyryl-CoA dehydrogenase (Bcd) of Acidaminococcus fermentans. J. Biol. Chem. 289, 5145–5157 (2014).

    Article  CAS  Google Scholar 

  11. Li, F. et al. Coupled ferredoxin and crotonyl coenzyme A (CoA) reduction with NADH catalyzed by the butyryl-CoA dehydrogenase/Etf complex from Clostridium kluyveri. J. Bacteriol. 190, 843–850 (2008).

    Article  CAS  Google Scholar 

  12. Chowdhury, N.P., Kahnt, J. & Buckel, W. Reduction of ferredoxin or oxygen by flavin-based electron bifurcation in Megasphaera elsdenii. FEBS J. 282, 3149–3160 (2015).

    Article  CAS  Google Scholar 

  13. Schut, G.J. & Adams, M.W.W. The iron-hydrogenase of Thermotoga maritima utilizes ferredoxin and NADH synergistically: a new perspective on anaerobic hydrogen production. J. Bacteriol. 191, 4451–4457 (2009).

    Article  CAS  Google Scholar 

  14. Brandt, U. Energy conservation by bifurcated electron-transfer in the cytochrome-bc 1 complex. Biochim. Biophys. Acta 1275, 41–46 (1996).

    Article  Google Scholar 

  15. Ptushenko, V.V., Cherepanov, D.A., Krishtalik, L.I. & Semenov, A.Y. Semi-continuum electrostatic calculations of redox potentials in photosystem I. Photosynth. Res. 97, 55–74 (2008).

    Article  CAS  Google Scholar 

  16. Hagen, W.R. et al. Novel structure and redox chemistry of the prosthetic groups of the iron-sulfur flavoprotein sulfide dehydrogenase from Pyrococcus furiosus; evidence for a [2Fe-2S] cluster with Asp(Cys)3 ligands. J. Biol. Inorg. Chem. 5, 527–534 (2000).

    Article  CAS  Google Scholar 

  17. Mohsen, A.W., Rigby, S.E.J., Jensen, K.F., Munro, A.W. & Scrutton, N.S. Thermodynamic basis of electron transfer in dihydroorotate dehydrogenase B from Lactococcus lactis: analysis by potentiometry, EPR spectroscopy, and ENDOR spectroscopy. Biochemistry 43, 6498–6510 (2004).

    Article  CAS  Google Scholar 

  18. Brereton, P.S., Verhagen, M.F.J.M., Zhou, Z.H. & Adams, M.W. Effect of iron-sulfur cluster environment in modulating the thermodynamic properties and biological function of ferredoxin from Pyrococcus furiosus. Biochemistry 37, 7351–7362 (1998).

    Article  CAS  Google Scholar 

  19. Hagen, W.R. Biomolecular EPR Spectroscopy (CRC press, 2008).

  20. Mathews, R., Charlton, S., Sands, R.H. & Palmer, G. On the nature of the spin coupling between the iron–sulfur clusters in the eight-iron ferredoxins. J. Biol. Chem. 249, 4326–4328 (1974).

    CAS  PubMed  Google Scholar 

  21. Anderson, R.F. Energetics of the one-electron reduction steps of riboflavin, FMN and FAD to their fully reduced forms. Biochim. Biophys. Acta 722, 158–162 (1983).

    Article  CAS  Google Scholar 

  22. Crofts, A.R. The cytochrome bc 1 complex: function in the context of structure. Annu. Rev. Physiol. 66, 689–733 (2004).

    Article  CAS  Google Scholar 

  23. Page, C.C., Moser, C.C., Chen, X. & Dutton, P.L. Natural engineering principles of electron tunnelling in biological oxidation–reduction. Nature 402, 47–52 (1999).

    Article  CAS  Google Scholar 

  24. Nitschke, W. & Russell, M.J. Redox bifurcations: mechanisms and importance to life now, and at its origin: a widespread means of energy conversion in biology unfolds.... BioEssays 34, 106–109 (2012).

    Article  CAS  Google Scholar 

  25. Crofts, A.R. & Rose, S. Marcus treatment of endergonic reactions: a commentary. Biochim. Biophys. Acta 1767, 1228–1232 (2007).

    Article  CAS  Google Scholar 

  26. Huang, H., Wang, S., Moll, J. & Thauer, R.K. Electron bifurcation involved in the energy metabolism of the acetogenic bacterium Moorella thermoacetica growing on glucose or H2 plus CO2 . J. Bacteriol. 194, 3689–3699 (2012).

    Article  CAS  Google Scholar 

  27. Keller, M.W. et al. Exploiting microbial hyperthermophilicity to produce an industrial chemical, using hydrogen and carbon dioxide. Proc. Natl. Acad. Sci. USA 110, 5840–5845 (2013).

    Article  CAS  Google Scholar 

  28. Horton, R.M., Hunt, H.D., Ho, S.N., Pullen, J.K. & Pease, L.R. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77, 61–68 (1989).

    Article  CAS  Google Scholar 

  29. Lipscomb, G.L. et al. Natural competence in the hyperthermophilic archaeon Pyrococcus furiosus facilitates genetic manipulation: construction of markerless deletions of genes encoding the two cytoplasmic hydrogenases. Appl. Environ. Microbiol. 77, 2232–2238 (2011).

    Article  CAS  Google Scholar 

  30. Verhagen, M.F., O'Rourke, T.W., Menon, A.L. & Adams, M.W. Heterologous expression and properties of the γ-subunit of the Fe-only hydrogenase from Thermotoga maritima. Biochim. Biophys. Acta 1505, 209–219 (2001).

    Article  CAS  Google Scholar 

  31. Aono, S., Bryant, F.O. & Adams, M.W. A novel and remarkably thermostable ferredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus. J. Bacteriol. 171, 3433–3439 (1989).

    Article  CAS  Google Scholar 

  32. Fourmond, V. QSoas: a versatile software for data analysis. Anal. Chem. 88, 5050–5052 (2016).

    Article  CAS  Google Scholar 

  33. Zehnder, A.J.B. & Wuhrmann, K. Titanium (III) citrate as a nontoxic oxidation-reduction buffering system for the culture of obligate anaerobes. Science 194, 1165–1166 (1976).

    Article  CAS  Google Scholar 

  34. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

  35. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  Google Scholar 

  36. Terwilliger, T.C. et al. Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard. Acta Crystallogr. D Biol. Crystallogr. 64, 61–69 (2008).

    Article  CAS  Google Scholar 

  37. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  38. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  39. DeLano, W.L. The PyMOL Molecular Graphics System (DeLano Scientific LLC, 2002).

  40. Baspinar, A., Cukuroglu, E., Nussinov, R., Keskin, O. & Gursoy, A. PRISM: a web server and repository for prediction of protein-protein interactions and modeling their 3D complexes. Nucleic Acids Res. 42, W285–W289 (2014).

    Article  CAS  Google Scholar 

  41. Comeau, S.R., Gatchell, D.W., Vajda, S. & Camacho, C.J. ClusPro: an automated docking and discrimination method for the prediction of protein complexes. Bioinformatics 20, 45–50 (2004).

    Article  CAS  Google Scholar 

  42. Garzon, J.I. et al. FRODOCK: a new approach for fast rotational protein-protein docking. Bioinformatics 25, 2544–2551 (2009).

    Article  CAS  Google Scholar 

  43. Massey, V. in Flavins and Flavoproteins (eds. Curti, B., Ronichi, S. & Zanetti, G.) 59–66 (Walter de Gruyter & Co., Berlin, 1991).

  44. Stoll, S. & Schweiger, A. EasySpin, a comprehensive software package for spectral simulation and analysis in EPR. J. Magn. Reson. 178, 42–55 (2006).

    Article  CAS  Google Scholar 

  45. Johnson, J.L., London, R.E. & Rajagopalan, K.V. Covalently bound phosphate residues in bovine milk xanthine oxidase and in glucose oxidase from Aspergillus niger: a reevaluation. Proc. Natl. Acad. Sci. USA 86, 6493–6497 (1989).

    Article  CAS  Google Scholar 

  46. Aliverti, A., Curti, B. & Vanoni, M.A. Identifying and quantitating FAD and FMN in simple and in iron–sulfur-containing flavoproteins. Methods Mol. Biol. 131, 9–23 (1999).

    CAS  PubMed  Google Scholar 

  47. Enescu, M., Lindqvist, L. & Soep, B. Excited-state dynamics of fully reduced flavins and flavoenzymes studied at subpicosecond time resolution. Photochem. Photobiol. 68, 150–156 (1998).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work and all authors were solely supported as part of the Biological and Electron Transfer and Catalysis (BETCy) EFRC, an Energy Frontier Research Center funded by the US Department of Energy, Office of Science, Basic Energy Sciences under Award # DE-SC0012518. Use of the Stanford Synchrotron Radiation Lightsource (SSRL), SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract no. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the National Institutes of Health, National Institute of General Medical Sciences (including P41GM103393). The Proteomics, Metabolomics, and Mass Spectrometry facility at MSU received support from the Murdock Charitable Trust and NIH 5P20RR02437 of the COBRE program. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the NIGMS or the NIH. C.E.L., D.W.M., and P.W.K. were supported by the US Department of Energy under contract no. DE-AC36-08-GO28308 with the National Renewable Energy Laboratory.

Author information

Authors and Affiliations

  1. Biosciences Center, National Renewable Energy Laboratory, Golden, Colorado, USA

    Carolyn E Lubner, David W Mulder & Paul W King

  2. School of Molecular Sciences, Arizona State University, Tempe, Arizona, USA

    David P Jennings & Anne K Jones

  3. Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA

    Gerrit J Schut, Diep M Nguyen, Gina L Lipscomb & Michael W W Adams

  4. Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana, USA

    Oleg A Zadvornyy, Monika Tokmina-Lukaszewska, Luke Berry, Brian Bothner & John W Peters

  5. Department of Chemistry, University of Kentucky, Lexington, Kentucky, USA

    John P Hoben & Anne-Frances Miller

  6. Institute of Biological Chemistry, Washington State University, Pullman, Washington, USA

    John W Peters

Authors
  1. Carolyn E Lubner
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David P Jennings
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. David W Mulder
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gerrit J Schut
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Oleg A Zadvornyy
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. John P Hoben
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Monika Tokmina-Lukaszewska
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Luke Berry
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Diep M Nguyen
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Gina L Lipscomb
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Brian Bothner
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Anne K Jones
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Anne-Frances Miller
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Paul W King
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Michael W W Adams
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. John W Peters
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.E.L. performed transient absorption spectroscopy experiments, analysis and interpretation; D.P.J. performed square wave voltammetry measurements, analysis and interpretation; D.W.M. performed EPR experiments, analysis and interpretation; G.J.S. generated and purified Pf NfnI protein, performed and interpreted redox titration, and developed and performed enzymatic assays; O.A.Z. performed structural characterization of Pf NfnI and analysis and interpretation; J.P.H. performed spectroelectrochemical titrations with guidance from A.-F.M.; M.T.-L. performed intact protein MS analysis; L.B. performed HDX–MS experiments, analysis and interpretation; D.M.N. and G.L.L. constructed Pf expression strains; B.B. contributed to HDX–MS analysis; all the authors conceived and designed the study; C.E.L., D.P.J., D.W.M., G.J.S., O.A.Z., B.B., A.K.J., A.-F.M., P.W.K., M.W.W.A. and J.W.P. contributed to the writing of the manuscript. C.E.L., D.P.J., D.W.M., G.J.S., and O.A.Z. contributed equally to the study.

Corresponding author

Correspondence to John W Peters.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–4 and Supplementary Figures 1–9 (PDF 9631 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lubner, C., Jennings, D., Mulder, D. et al. Mechanistic insights into energy conservation by flavin-based electron bifurcation. Nat Chem Biol 13, 655–659 (2017). https://doi.org/10.1038/nchembio.2348

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.2348

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing