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:

Proton-powered subunit rotation in single membrane-bound F0F1-ATP synthase

Nature Structural & Molecular Biology volume 11, pages 135–141 (2004)Cite this article

Abstract

Synthesis of ATP from ADP and phosphate, catalyzed by F0F1-ATP synthases, is the most abundant physiological reaction in almost any cell. F0F1-ATP synthases are membrane-bound enzymes that use the energy derived from an electrochemical proton gradient for ATP formation. We incorporated double-labeled F0F1-ATP synthases from Escherichia coli into liposomes and measured single-molecule fluorescence resonance energy transfer (FRET) during ATP synthesis and hydrolysis. The γ subunit rotates stepwise during proton transport–powered ATP synthesis, showing three distinct distances to the b subunits in repeating sequences. The average durations of these steps correspond to catalytic turnover times upon ATP synthesis as well as ATP hydrolysis. The direction of rotation during ATP synthesis is opposite to that of ATP hydrolysis.

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: Model of F0F1 from E. coli (see Methods).
Figure 2: Photon bursts from single F0F1-ATP synthases in liposomes.
Figure 3: Photon bursts from single liposomes with one double-labeled F0F1-ATP synthase in the presence of 1 mM AMPPNP.
Figure 4: Histograms of the fluorescence intensity ratios FD / FA of single FRET-labeled F0F1.
Figure 5: Level duration distributions of FRET states.

Similar content being viewed by others

References

  1. Mitchell, P. Coupling of phosphorylation to electron and hydrogen transfer by chemi-osmotic type of mechanism. Nature 191, 144–152 (1961).

    Article  CAS  Google Scholar 

  2. Boyer, P.D. ATP synthase—past and future. Biochim. Biophys. Acta 1365, 3–9 (1998).

    Article  CAS  Google Scholar 

  3. Abrahams, J.P., Leslie, A.G.W., Lutter, R. & Walker, J.E. Structure at 2.8 Å resolution of F1-ATPase from bovine heart mitochondria. Nature 370, 621–628 (1994).

    Article  CAS  Google Scholar 

  4. Junge, W., Sabbert, D. & Engelbrecht, S. Rotatory catalysis by F-ATPase: Real-time recording of intersubunit rotation. Ber. Bunsenges. Phys. Chem. 100, 2014–2019 (1996).

    Article  CAS  Google Scholar 

  5. Yoshida, M., Muneyuki, E. & Hisabori, T. ATP synthase – a marvelous rotary engine of the cell. Nat. Rev. Mol. Cell. Biol. 2, 669–677 (2001).

    Article  CAS  Google Scholar 

  6. Capaldi, R.A. & Aggeler, R. Mechanism of the F1F0-type ATP synthase, a biological rotary motor. Trends Biochem. Sci. 27, 154–160 (2002).

    Article  CAS  Google Scholar 

  7. Weber, J. & Senior, A.E. ATP synthesis driven by proton transport in F1F0-ATP synthase. FEBS Lett. 545, 61–70 (2003).

    Article  CAS  Google Scholar 

  8. Fillingame, R.H., Angevine, C.M. & Dmitriev, O.Y. Coupling proton movements to c-ring rotation in F1F0 ATP synthase: aqueous access channels and helix rotations at the a-c interface. Biochim. Biophys. Acta 1555, 29–36 (2002).

    Article  CAS  Google Scholar 

  9. Noji, H., Yasuda, R., Yoshida, M. & Kinosita, K. Jr. Direct observation of the rotation of F1-ATPase. Nature 386, 299–302 (1997).

    Article  CAS  Google Scholar 

  10. Yasuda, R., Noji, H., Yoshida, M., Kinosita, K. & Itoh, H. Resolution of distinct rotational sub-steps by submillisecond kinetic analysis of F1-ATPase. Nature 410, 898–904 (2001).

    Article  CAS  Google Scholar 

  11. Yasuda, R. et al. The ATP-waiting conformation of rotating F1-ATPase revealed by single-pair fluorescence resonance energy transfer. Proc. Natl. Acad. Sci. USA 100, 9314–9318 (2003).

    Article  CAS  Google Scholar 

  12. Sambongi, Y. et al. Mechanical rotation of the c subunit oligomer in ATP synthase (F0F1): direct observation. Science 286, 1722–1724 (1999).

    Article  CAS  Google Scholar 

  13. Junge, W. et al. Inter-subunit rotation and elastic power transmission in F0F1-ATPase. FEBS Lett. 504, 152–160 (2001).

    Article  CAS  Google Scholar 

  14. Kaim, G. et al. Coupled rotation within single F0F1 enzyme complexes during ATP synthesis or hydrolysis. FEBS Lett. 525, 156–163 (2002).

    Article  CAS  Google Scholar 

  15. Böckmann, R.A. & Grubmüller, H. Nanosecond molecular dynamics simulation of primary mechanical energy transfer steps in F1-ATP synthase. Nat. Struct. Biol. 9, 198–202 (2002).

    PubMed  Google Scholar 

  16. Ma, J. et al. A dynamical analysis of the rotation mechanism for conformational change in F1-ATPase. Structure 10, 921–930 (2002).

    Article  CAS  Google Scholar 

  17. Zhou, Y., Duncan, T.M., Cross, R.L. Subunit rotation in Escherichia coli F0F1-ATP synthase during oxidative phosphorylation. Proc. Natl. Acad. Sci. USA 94, 10583–10587 (1997).

    Article  CAS  Google Scholar 

  18. Börsch, M., Diez, M., Zimmermann, B., Reuter, R. & Gräber, P. Stepwise rotation of the γ-subunit of EF0F1-ATP synthase observed by intramolecular single-molecule fluorescence resonance energy transfer. FEBS Lett 527, 147–152 (2002).

    Article  Google Scholar 

  19. Fischer, S. & Gräber, P. Comparison of ΔpH- and Δφ-driven ATP synthesis catalyzed by the H+-ATPases from Escherichia coli or chloroplasts reconstituted into liposomes. FEBS Lett. 457, 327–332 (1999).

    Article  CAS  Google Scholar 

  20. Fischer, S., Gräber, P. & Turina, P. The activity of the ATP synthase from Escherichia coli is regulated by the transmembrane proton motive force. J. Biol. Chem. 275, 30157–30162 (2000).

    Article  CAS  Google Scholar 

  21. Lötscher, H.R. deJong, C. & Capaldi, R.A. Modification of the F0 portion of the H+-translocating adenosinetriphosphatase complex of Escherichia coli by the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and effect on the proton channeling function. Biochemistry 23, 4128–4134 (1983).

    Article  Google Scholar 

  22. Perlin, D.S., Cox, D.N. & Senior, A.E. Integration of F1 and the membrane sector of the proton-ATPase of E coli. J. Biol. Chem. 258, 9793–9800 (1983).

    CAS  PubMed  Google Scholar 

  23. Weiss, S. Measuring conformational dynamics of biomolecules by single molecule fluorescence spectroscopy. Nature Struct. Biol. 7, 724–729 (2000).

    Article  CAS  Google Scholar 

  24. Ha, T. Single-molecule fluorescence resonance energy transfer. Methods 25, 78–86 (2001).

    Article  CAS  Google Scholar 

  25. Rothwell, P.J. et al. Multi-parameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase: primer/template complexes. Proc. Natl. Acad. Sci. USA 100, 1655–1660 (2003).

    Article  CAS  Google Scholar 

  26. Förster, T. Zwischenmolekulare Energiewanderung und Fluoreszenz. Ann. Phys. 2, 55–70 (1948).

    Article  Google Scholar 

  27. Van der Meer, B.W., Cooker, G. & Chen, S.S.-Y. Resonance Energy Transfer: Theory and Data (VCH, New York, 1994).

    Google Scholar 

  28. Häsler, K., Engelbrecht, S. & Junge, W. Three-stepped rotation of subunits Q and O in single molecules of F-ATPase as revealed by polarized, confocal fluorometry. FEBS Lett. 426, 301–304 (1998).

    Article  Google Scholar 

  29. Gogol, E.P., Luecken, U., Bork, T. & Capaldi, R.A. Molecular architecture of Escherichia coli F1 adenosinetriphosphatase. Biochemistry 28, 4709–4716 (1989).

    Article  CAS  Google Scholar 

  30. Aggeler, R. & Capaldi, R.A. Cross-linking of the γ subunit of the Escherichia coli ATPase (ECF1) via cysteines introduced by site-directed mutagenesis. J. Biol. Chem. 267, 21355–21359 (1992).

    CAS  PubMed  Google Scholar 

  31. Aggeler, R., Ogilvie, I. & Capaldi, R.A. Rotation of a γ-ε subunit domain in the Escherichia coli F1F0-ATP synthase complex. J. Biol. Chem. 272, 19621–19624 (1997).

    Article  CAS  Google Scholar 

  32. Börsch, M. et al. Conformational changes of the H+-ATPase from Escherichia coli upon nucleotide binding detected by single molecule fluorescence. FEBS Lett. 437, 251–254 (1998).

    Article  Google Scholar 

  33. Turina, P. & Capaldi, R.A. ATP hydrolysis-driven structural changes in the γ-subunit of Escherichia coli ATPase monitored by fluorescence from probes bound at introduced cysteine residues. J. Biol. Chem. 269 13465–13471 (1994).

    CAS  PubMed  Google Scholar 

  34. Engelbrecht, S. & Junge, W. ATP synthase: a tentative structural model. FEBS Lett 414, 485–491 (1997).

    Article  CAS  Google Scholar 

  35. Rodgers, A. & Wilce, M. Structure of the γ/ε complex of ATP synthase: the camshaft in the rotary motor of life. Nat. Struct. Biol. 7, 1051–1054 (2000).

    Article  CAS  Google Scholar 

  36. Wilkens, S., Dunn, S., Chandler, J., Dahlquist, F.W. & Capaldi, R.A. Solution structure of the N-terminal domain of the δ subunit of the E. coli ATP synthase (ECF1F0). Nat. Struct. Biol. 4, 198–201 (1997).

    Article  CAS  Google Scholar 

  37. Girvin, M.E., Rastogi, V.K., Abildgaard, F., Markley, J.L. & Fillingame, R.H. Solution structure of the transmembrane H+-transporting subunit c of the F0F1 ATP synthase. Biochemistry. 37, 8817–8824 (1998).

    Article  CAS  Google Scholar 

  38. Del Rizzo, P.A., Dunn, S.D., Bi, Y. & Shilton, B.H. The 'second stalk' of Escherichia coli ATP synthase: structure of the isolated dimerization domain. Biochemistry 41, 6875–6884 (2002).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work is dedicated to the memory of K. Süss, who died on 17 March 2002. We thank R.H. Fillingame for his help with the b-mutants, R.A. Capaldi and R. Aggeler for the gift of the γ-mutant, O. Hucke for refinement of the F0F1 model, and M. Antonik and E. Haustein for analytical software. We thank H. Grubmüller, R. Jahn, B.A. Melandri and P. Turina for critical reading the manuscript and helpful discussions and A. Börsch-Haubold for editorial suggestions. C.A.M.S. acknowledges financial support by the Bundesministerium für Bildung und Forschung (BioFuture grant 0311865).

Author information

Authors and Affiliations

  1. Institut für Physikalische Chemie, Albert-Ludwigs-Universität Freiburg, Albertstrasse 23 a, Freiburg, 79104, Germany

    Manuel Diez, Boris Zimmermann, Stefan Steigmiller, Rolf Reuter & Peter Gräber

  2. 3. Physikalisches Institut, Universität Stuttgart, Pfaffenwaldring 57, Stuttgart, 70569, Germany

    Michael Börsch

  3. Max-Planck-Institut für Biophysikalische Chemie, Am Fassberg 11, Göttingen, 37077, Germany

    Marcelle König, Enno Schweinberger, Suren Felekyan, Volodymyr Kudryavtsev & Claus A M Seidel

Authors
  1. Manuel Diez
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Boris Zimmermann
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michael Börsch
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Marcelle König
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Enno Schweinberger
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Stefan Steigmiller
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Rolf Reuter
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Suren Felekyan
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Volodymyr Kudryavtsev
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Claus A M Seidel
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Peter Gräber
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Michael Börsch.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Diez, M., Zimmermann, B., Börsch, M. et al. Proton-powered subunit rotation in single membrane-bound F0F1-ATP synthase. Nat Struct Mol Biol 11, 135–141 (2004). https://doi.org/10.1038/nsmb718

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb718

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