Highly coupled ATP synthesis by F1-ATPase single molecules

F 1 -ATPase is the smallest known rotary motor, and it rotates in an anticlockwise direction as it hydrolyses ATP 1 , 2 , 3 , 4 , 5 . Single-molecule experiments 6 , 7 , 8 , 9 point towards three catalytic events per turn, in agreement with the molecular structure of the complex 10 . The physiological function of F 1 is ATP synthesis. In the ubiquitous F 0 F 1 complex, this energetically uphill reaction is driven by F 0 , the partner motor of F 1 , which forces the backward (clockwise) rotation of F 1 , leading to ATP synthesis 11 , 12 , 13 . Here, we have devised an experiment combining single-molecule manipulation and microfabrication techniques to measure the yield of this mechanochemical transformation. Single F 1 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 F 1 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 α 3 β 3 γ subcomplex (F 1 -ɛ ), but reached up to 77% after reconstitution with the ɛ-subunit (F 1 +ɛ ). We provide here direct evidence that F 1 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.