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Highly coupled ATP synthesis by F1-ATPase single molecules

Nature volume 433, pages 773–777 (2005)Cite this article

Abstract

F1-ATPase is the smallest known rotary motor, and it rotates in an anticlockwise direction as it hydrolyses ATP1,2,3,4,5. Single-molecule experiments6,7,8,9 point towards three catalytic events per turn, in agreement with the molecular structure of the complex10. The physiological function of F1 is ATP synthesis. In the ubiquitous F0F1 complex, this energetically uphill reaction is driven by F0, the partner motor of F1, which forces the backward (clockwise) rotation of F1, leading to ATP synthesis11,12,13. Here, we have devised an experiment combining single-molecule manipulation and microfabrication techniques to measure the yield of this mechanochemical transformation. Single F1 molecules were enclosed in femtolitre-sized hermetic chambers and rotated in a clockwise direction using magnetic tweezers. When the magnetic field was switched off, the F1 molecule underwent anticlockwise rotation at a speed proportional to the amount of synthesized ATP. At 10 Hz, the mechanochemical coupling efficiency was low for the α3β3γ subcomplex (F1-ɛ), but reached up to 77% after reconstitution with the ɛ-subunit (F1+ɛ). We provide here direct evidence that F1 is designed to tightly couple its catalytic reactions with the mechanical rotation. Our results suggest that the ɛ-subunit has an essential function during ATP synthesis.

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Figure 1: ATP synthesis by F1-ATPase.
Figure 2: Determination of the coupling efficiency of ATP hydrolysis.
Figure 3: Determination of the coupling efficiency of ATP synthesis.

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Acknowledgements

We thank all members of the Noji and Takeuchi laboratories, and H. Arata and A. Tixier-Mita for discussion and experimental support; R. Yasuda for PC programming of image analysis (CREST image); and Central Workshop in IIS for an optical microscope stage. This work was performed in the framework of LIMMS/CNRS-IIS, and supported in part by Bio-oriented Technology Research Advancement Institution (H.N. and S.T.), and Grants-in-Aid from Ministry of Education, Science, Sports and Culture of Japan (H.N., H.F. and S.T.). Y.R. and G.T. are Research Fellows of the Japan Society for the Promotion of Science.

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Authors and Affiliations

  1. LIMMS/CNRS-IIS,

    Yannick Rondelez & Guillaume Tresset

  2. Institute of Industrial Science, The University of Tokyo, 153-8505, Tokyo, Japan

    Yannick Rondelez, Guillaume Tresset, Takako Nakashima & Hiroyuki Noji

  3. Department of Life Science, College of Science, Rikkyo (St Paul's) University, 171-8501, Tokyo, Japan

    Yasuyuki Kato-Yamada

  4. CIRMM, Institute of Industrial Science, The University of Tokyo, 153-8505, Tokyo, Japan

    Hiroyuki Fujita & Shoji Takeuchi

Authors
  1. Yannick Rondelez
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  2. Guillaume Tresset
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  3. Takako Nakashima
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  4. Yasuyuki Kato-Yamada
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  5. Hiroyuki Fujita
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  6. Shoji Takeuchi
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  7. Hiroyuki Noji
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Correspondence to Hiroyuki Noji.

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The authors declare that they have no competing financial interests.

Supplementary information

Video of an ATP synthesis experiment (AVI 17267 kb)

Supplemental Information

1. The video time table for the ATP synthesis experiment. 2. Standard curves of rotational velocity versus ATP concentration. (DOC 38 kb)

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Rondelez, Y., Tresset, G., Nakashima, T. et al. Highly coupled ATP synthesis by F1-ATPase single molecules. Nature 433, 773–777 (2005). https://doi.org/10.1038/nature03277

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