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(1-5). Single-molecule experiments(6-9) 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 F0F1 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(11-13). 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 alpha(3)beta(3)gamma subcomplex (F-1(-epsilon)), but reached up to 77% after reconstitution with the epsilon-subunit (F-1(+epsilon)). 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 epsilon-subunit has an essential function during ATP synthesis.