Highly coupled ATP synthesis by F1-ATPase single molecules.
F1-ATPase is the smallest known rotary motor, and it rotates in an anticlockwise direction as it hydrolyses ATP. Single-molecule experiments point towards three catalytic events per turn, in agreement with the molecular structure of the complex. 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 synthesis. 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 alpha3beta3gamma subcomplex (F1-epsilon)), but reached up to 77% after reconstitution with the epsilon-subunit (F1+epsilon)). We provide here direct evidence that F1 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.
Rondelez
Yannick
Y
LIMMS/CNRS-IIS, Tokyo 153-8505, Japan.
Tresset
Guillaume
G
Nakashima
Takako
T
Kato-Yamada
Yasuyuki
Y
Fujita
Hiroyuki
H
Takeuchi
Shoji
S
Noji
Hiroyuki
H
D000255:Adenosine Triphosphate / Q000096:biosynthesis* / Q000378:metabolism; D001696:Biomechanical Phenomena; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012399:Rotation; D013997:Time Factors
D004800:Enzymes, Immobilized; D020409:Molecular Motor Proteins; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases