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:

Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase

Nature volume 410, pages 898–904 (2001)Cite this article

Abstract

The enzyme F1-ATPase has been shown to be a rotary motor in which the central γ-subunit rotates inside the cylinder made of α3β3 subunits. At low ATP concentrations, the motor rotates in discrete 120° steps, consistent with sequential ATP hydrolysis on the three β-subunits. The mechanism of stepping is unknown. Here we show by high-speed imaging that the 120° step consists of roughly 90° and 30° substeps, each taking only a fraction of a millisecond. ATP binding drives the 90° substep, and the 30° substep is probably driven by release of a hydrolysis product. The two substeps are separated by two reactions of about 1 ms, which together occupy most of the ATP hydrolysis cycle. This scheme probably applies to rotation at full speed (∼130 revolutions per second at saturating ATP) down to occasional stepping at nanomolar ATP concentrations, and supports the binding-change model for ATP synthesis by reverse rotation of F1-ATPase.

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: Observation of F1 rotation.
Figure 2: Relationship between rate of bead rotation and viscous friction on the bead.
Figure 3: Comparison of rotation and hydrolysis rates.
Figure 4: Unfiltered time courses of stepping rotation of 40-nm beads at varying [ATP].
Figure 5: Histograms of angular positions over 0.5 s runs.
Figure 6: Kinetics of substeps.
Figure 7: Proposed mechanism for F1 rotation.
Figure 8: Dwells between main steps.

Similar content being viewed by others

References

  1. Boyer, P. D. & Kohlbrenner, W. in Energy Coupling in Photosynthesis (eds Selman, B. R. & Selman-Reimer, S.) 231–240 (Elsevier, Amsterdam, 1981).

    Google Scholar 

  2. Boyer, P. D. The binding change mechanism for ATP synthase—some probabilities and possibilities. Biochim. Biophys. Acta 1140, 215–250 (1993).

    Article  ADS  CAS  Google Scholar 

  3. Boyer, P. D. Catalytic site forms and controls in ATP synthase catalysis. Biochim. Biophys. Acta 1458, 252–262 (2000).

    Article  CAS  Google Scholar 

  4. Cox, G. B., Jans, D. A., Fimmel, A. L., Gibson, F. & Hatch, L. The mechanism of ATP synthase. Conformational change by rotation of the b-subunit. Biochim. Biophys. Acta 768, 201–208 (1984).

    Article  CAS  Google Scholar 

  5. Mitchell, P. Molecular mechanics of protonmotive FoF1 ATPases. Rolling well and turnstile hypothesis. FEBS Lett. 182, 1–7 (1985).

    Article  CAS  Google Scholar 

  6. Oosawa, F. & Hayashi, S. The loose coupling mechanism in molecular machines of living cells. Adv. Biophys. 22, 151–183 (1986).

    Article  CAS  Google Scholar 

  7. 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  ADS  CAS  Google Scholar 

  8. Duncan, T. M., Bulygin, V. V., Zhou, Y., Hutcheon, M. L. & Cross, R. Rotation of subunits during catalysis by Escherichia coli F1-ATPase. Proc. Natl Acad. Sci. USA 92, 10964–10968 (1995).

    Article  ADS  CAS  Google Scholar 

  9. Sabbert, D., Engelbrecht, S. & Junge, W. Intersubunit rotation in active F-ATPase. Nature 381, 623–625 (1996).

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  11. Yasuda, R., Noji, H., Kinosita, K. Jr & Yoshida, M. F1-ATPase is a highly efficient molecular motor that rotates with discrete 120° steps. Cell 93, 1117–1124 (1998).

    Article  CAS  Google Scholar 

  12. Kudo, S., Magariyama, Y. & Aizawa, S. Abrupt changes in flagellar rotation observed by laser dark-field microscopy. Nature 346, 677–680 (1990).

    Article  ADS  CAS  Google Scholar 

  13. Miyata, H. et al. Stepwise motion of an actin filament over a small number of heavy meromyosin molecules is revealed in an in vitro motility assay. J. Biochem. (Tokyo) 115, 644–647 (1994).

    Article  CAS  Google Scholar 

  14. Weber, P. C., Ohlendorf, D. H., Wendoloski, J. J. & Salemme, F. R. Structural origins of high-affinity biotin binding to streptavidin. Science 243, 85–88 (1989).

    Article  ADS  CAS  Google Scholar 

  15. He, X. M. & Carter, D. C. Atomic structure and chemistry of human serum albumin. Nature 358, 209–215 (1992).

    Article  ADS  CAS  Google Scholar 

  16. Kinosita, K. Jr, Yasuda, R. & Noji, H. F1-ATPase: a highly efficient rotary ATP machine. Essays Biochem. 35, 3–18 (2000).

    Article  CAS  Google Scholar 

  17. Kinosita, K. Jr, Yasuda, R., Noji, H. & Adachi, K. A rotary molecular motor that can work at near 100% efficiency. Phil. Trans. R. Soc. Lond. B 355, 473–489 (2000).

    Article  CAS  Google Scholar 

  18. Adachi, K. et al. Stepping rotation of F1-ATPase visualized through angle-resolved single-fluorophore imaging. Proc. Natl Acad. Sci. USA 97, 7243–7247 (2000).

    Article  ADS  CAS  Google Scholar 

  19. Jault, J.-M. et al. The α3β3γ complex of the F1-ATPase from the thermophilic Bacillus PS3 containing the αD261N substitution fails to dissociate inhibitory Mg ADP from a catalytic site when ATP binds to noncatalytic sites. Biochemistry 34, 16412–16418 (1995).

    Article  CAS  Google Scholar 

  20. Matsui, T. et al. Catalytic activity of the α3β3γ complex of F1-ATPase without noncatalytic nucleotide binding site. J. Biol. Chem. 272, 8215–8221 (1997).

    Article  CAS  Google Scholar 

  21. Cunningham, D. & Cross, R. L. Catalytic site occupancy during ATP hydrolysis by MF1-ATPase. Evidence for alternating high affinity sites during steady-state turnover. J. Biol. Chem. 263, 18850–18565 (1988).

    CAS  PubMed  Google Scholar 

  22. Gresser, M. J., Myers, J. A. & Boyer, P. D. Catalytic site cooperativity of beef heart mitochondrial F1 adenosine triphosphatase. Correlations of initial velocity, bound intermediate, and oxygen exchange measurements with an alternating three-site model. J. Biol. Chem. 257, 12030–12038 (1982).

    CAS  PubMed  Google Scholar 

  23. Jault, J.-M. et al. The α3β3γ subcomplex of the F1-ATPase from the thermophilic Bacillus PS3 with the βT165S substitution does not entrap inhibitory MgADP in a catalytic site during turnover. J. Biol. Chem. 271, 28818–28824 (1996).

    Article  CAS  Google Scholar 

  24. Weber, J., Wilke-Mounts, S., Lee, R. S., Grell, E. & Senior, A. E. Specific placement of tryptophan in the catalytic sites of Escherichia coli F1-ATPase provides a direct probe of nucleotide binding: maximal ATP hydrolysis occurs with three sites occupied. J. Biol. Chem. 268, 20126–20133 (1993).

    CAS  PubMed  Google Scholar 

  25. Milgrom, Y. M., Murataliev, M. B. & Boyer, P. D. Bi-site activation occurs with the native and nucleotide-depleted mitochondrial F1-ATPase. Biochem. J. 330, 1037–1043 (1998).

    Article  CAS  Google Scholar 

  26. Zhou, J.-M. & Boyer, P. D. Evidence that energization of the chloroplast ATP synthase favors ATP formation at the tight binding catalytic site and increases the affinity for ADP at another catalytic site. J. Biol. Chem. 268, 1531–1538 (1993).

    CAS  PubMed  Google Scholar 

  27. Gibbons, C., Montgomery, M. G., Leslie, A. G. W. & Walker, J. E. The structure of the central stalk in bovine F1-ATPase at 2.4 Å resolution. Nature Struct. Biol. 7, 1055–1061 (2000).

    Article  CAS  Google Scholar 

  28. Wang, H. & Oster, G. Energy transduction in the F1 motor of ATP synthase. Nature 396, 279–282 (1998).

    Article  ADS  CAS  Google Scholar 

  29. Wolcott, R. G. & Boyer, P. D. The reversal of the myosin and actomyosin ATPase reactions and the free energy of ATP binding to myosin. Biochem. Biophys. Res. Commun. 57, 709–716 (1974).

    Article  CAS  Google Scholar 

  30. Mannherz, H. G., Schenck, H. & Goody, R. S. Synthesis of ATP from ADP and inorganic phosphate at the myosin-subfragment 1 active site. Eur. J. Biochem. 48, 287–295 (1974).

    Article  CAS  Google Scholar 

  31. Houdusse, A., Szent-Györgyi, A. G. & Cohen, C. Three conformational states of scallop myosin S1. Proc. Natl Acad. Sci. USA 97, 11238–11243 (2000).

    Article  ADS  CAS  Google Scholar 

  32. Rice, S. et al. A structural change in the kinesin motor protein that drives motility. Nature 402, 778–784 (1999).

    Article  ADS  CAS  Google Scholar 

  33. Schnitzer, M. J., Visscher, K. & Block, S. M. Force production by single kinesin motors. Nature Cell Biol. 2, 718–723 (2000).

    Article  CAS  Google Scholar 

  34. Sigler, P. B. et al. Structure and function in GroEL-mediated protein folding. Annu. Rev. Biochem. 67, 581–608 (1998).

    Article  CAS  Google Scholar 

  35. Kunioka, Y. & Ando, T. Innocuous labeling of the subfragment-2 region of skeletal muscle heavy meromyosin with a fluorescent polyacrylamide nanobead and visualization of individual heavy meromyosin molecules. J. Biochem. (Tokyo) 119, 1024–1032 (1996).

    Article  CAS  Google Scholar 

  36. Kato, Y., Sasayama, T., Muneyuki, E. & Yoshida, M. Analysis of time-dependent change of Escherichia coli F1-ATPase activity and its relationship with apparent negative cooperativity. Biochim. Biophys. Acta 1231, 275–281 (1995).

    Article  Google Scholar 

  37. Born, M. & Wolf, E. Principles of Optics 7th edn. (Cambridge Univ. Press, Cambridge, 1999).

    Book  Google Scholar 

Download references

Acknowledgements

We thank T. Ariga for sample preparation; A. Kusumi for colloidal gold; T. Hisabori, E. Muneyuki, T. Nishizaka, K. Adachi, C. Gosse, M. Y. Ali, S. Ishiwata and G. W. Feigenson for critical discussions; and H. Umezawa for laboratory management. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan.

Author information

Author notes

  1. Ryohei Yasuda

    Present address: Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York, 11724, USA

Authors and Affiliations

  1. CREST ‘Genetic Programming’ Team 13, Teikyo University Biotechnology Center 3F, Nogawa 907, Miyamae-Ku, Kawasaki, 216-0001, Japan

    Ryohei Yasuda, Hiroyuki Noji, Masasuke Yoshida, Kazuhiko Kinosita Jr & Hiroyasu Itoh

  2. Department of Physics, Faculty of Science and Technology, Keio University, Yokohama, 223-8522, Japan

    Ryohei Yasuda & Kazuhiko Kinosita Jr

  3. Chemical Resources Laboratory, Tokyo Institute of Technology, Yokohama, 226-8503, Japan

    Masasuke Yoshida

  4. Tsukuba Research Laboratory, Hamamatsu Photonics KK, Tokodai, Tsukuba, 300-2635, Japan

    Hiroyasu Itoh

Authors
  1. Ryohei Yasuda
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hiroyuki Noji
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Masasuke Yoshida
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kazuhiko Kinosita Jr
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hiroyasu Itoh
    View author publications

    You can also search for this author in PubMed Google Scholar

Supplementary information

You will need a Quick Time Player to view the below movies.

Movie 1 (mov 1.7 mb)

Stepping rotation of a 40-nm bead attached to the gamma subunit of F1-ATPase. ATP concentration, 20 micromolar. Close look at the movie will reveal 90- and 30-degree substeps. Images were recorded at 8,000 frames per second and are played at 11 frames per second. Diameter of the circular images, 320nm.

Movie 2 (mov 1 mb)

Stepping rotation of a 40-nm bead attached to the gamma subunit of F1-ATPase. ATP concentration, 2 mM. Images were recorded at 8,000 frames per second and are played at 11 frames per second. Diameter of the circular images, 320nm.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yasuda, R., Noji, H., Yoshida, M. et al. Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase. Nature 410, 898–904 (2001). https://doi.org/10.1038/35073513

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/35073513

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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