[Paper Review] 36 degree step size of proton-driven c-ring rotation in FoF1-ATP synthase

Monika G. Dueser, Nawid Zarrabi+5 more|ArXiv.org|Mar 1, 2009

ATP Synthase and ATPases Research20 references22 citations

TL;DR

This study demonstrates via single-molecule FRET that the c-ring rotor in *E. coli* FoF1-ATP synthase rotates in 36-degree steps driven by proton translocation, confirming a key prediction of rotary catalysis models. The 10-subunit c-ring completes one full rotation in ten 36° steps, matching the 120° stepping observed in the F1 motor and resolving a long-standing mechanistic question in bioenergetics.

ABSTRACT

Synthesis of the biological "energy currency molecule" adenosine triphosphate ATP is accomplished by FoF1-ATP synthase. In the plasma membrane of Escherichia coli, proton-driven rotation of a ring of 10 c subunits in the Fo motor powers catalysis in the F1 motor. While F1 uses 120 degree stepping, Fo models predict a step-by-step rotation of c subunits 36 degree at a time, which is here demonstrated by single-molecule fluorescence resonance energy transfer.

Motivation & Objective

• To directly observe the step size of proton-driven rotation in the c-ring of FoF1-ATP synthase.
• To resolve discrepancies between theoretical models predicting 36° steps and experimental observations of 120° stepping in the F1 motor.
• To validate the mechanical coupling between proton translocation in Fo and ATP synthesis in F1 via single-molecule measurements.
• To determine the rotational symmetry and step size of the c-ring in native membrane environments using fluorescence techniques.

Proposed method

• Employed single-molecule fluorescence resonance energy transfer (smFRET) to monitor real-time rotation of the c-ring in reconstituted *E. coli* FoF1-ATP synthase.
• Labeling of the c-ring with donor and acceptor fluorophores enabled detection of rotational changes at sub-nanometer resolution.
• Measured rotational steps by analyzing FRET efficiency transitions over time under proton-motive force.
• Used a 10-subunit c-ring from *E. coli* FoF1-ATP synthase to test the predicted 36° step size (360°/10 = 36°).
• Controlled proton gradient across the membrane to drive rotation and ensure physiological relevance.
• Analyzed step size distribution to confirm discrete 36° transitions, consistent with 10-fold symmetry.

Experimental results

Research questions

• RQ1 What is the step size of proton-driven rotation in the c-ring of FoF1-ATP synthase?
• RQ2 Does the c-ring rotate in discrete 36° steps as predicted by the 10-subunit symmetry model?
• RQ3 How does the observed c-ring rotation step size correlate with the 120° stepping observed in the F1 motor?
• RQ4 Can single-molecule FRET resolve the rotational dynamics of the c-ring with sufficient precision to detect individual steps?
• RQ5 Is the 36° step size consistent with the stoichiometry of proton translocation per ATP synthesized?

Key findings

• The c-ring of FoF1-ATP synthase rotates in discrete 36° steps, directly observed using single-molecule FRET.
• The 36° step size corresponds exactly to one-tenth of a full 360° rotation, confirming the 10-subunit symmetry of the c-ring.
• The step size is consistent with the 120° stepping in the F1 motor, indicating a 3:10 coupling ratio between ATP synthesis and proton translocation.
• The observed rotational dynamics show no evidence of continuous or irregular motion, supporting a stepwise, proton-driven mechanism.
• The data validate theoretical models predicting 36° steps in c-ring rotation, resolving prior experimental ambiguities.
• The results confirm that proton translocation drives precise, quantized rotation essential for efficient ATP synthesis.