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Phosphate release in F1-ATPase catalytic cycle follows ADP release

Nature Chemical Biology volume 6, pages 814–820 (2010)Cite this article

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Abstract

F1-ATPase is an ATP-driven rotary motor protein in which the γ-subunit rotates against the catalytic stator ring. Although the reaction scheme of F1 has mostly been revealed, the timing of inorganic phosphate (Pi) release remains controversial. Here we addressed this issue by verifying the reversibility of ATP hydrolysis on arrested F1 with magnetic tweezers. ATP hydrolysis was found to be essentially reversible, implying that Pi is released after the γ rotation and ADP release, although extremely slow Pi release was found at the ATP hydrolysis angle as an uncoupling side reaction. On the basis of this finding, we deduced the chemomechanical coupling scheme of F1. We found that the affinity for Pi was strongly angle dependent, implying a large contribution by Pi release to torque generation. These findings imply that under ATP synthesis conditions, Pi binds to an empty catalytic site, preventing solution ATP (though not ADP) from binding. Thus, this supports the concept of selective ADP binding for efficient ATP synthesis.

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Figure 1: Models of Pi release in chemomechanical coupling of F1.
Figure 2: Reversibility of ATPγS hydrolysis and synthesis on F1(βE190D).
Figure 3: Reversibility of ATP hydrolysis and synthesis of β(E190D) in the hybrid F1.
Figure 4: Synthesis of ATPγS.
Figure 5: Equilibrium constant of Pi release and binding.
Figure 6: Site occupancy and reaction scheme.

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Acknowledgements

We thank K. Hayashi for critical discussion, H. Ueno for sample preparation and all members of Noji laboratory for technical support. This work was partially supported by the Grant-in-Aid for Scientific Research No. 18074005 to H.N. and by special education and research expenses (to H.N.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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Authors and Affiliations

  1. Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan

    Rikiya Watanabe, Ryota Iino & Hiroyuki Noji

  2. Department of Applied Chemistry, School of Engineering, The University of Tokyo, Tokyo, Japan

    Hiroyuki Noji

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R.W. designed and performed experiments and analyzed data; R.I. gave technical support and conceptual advice; and H.N. designed experiments and wrote the paper.

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Correspondence to Hiroyuki Noji.

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Watanabe, R., Iino, R. & Noji, H. Phosphate release in F1-ATPase catalytic cycle follows ADP release. Nat Chem Biol 6, 814–820 (2010). https://doi.org/10.1038/nchembio.443

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