Simultaneous Monitoring The Two Rotary Motors Of A Single FOF1-ATP Synthase

Abstract

Synthesis of ATP from ADP and phosphate is performed by a stepwise internal rotation of subunits of the enzyme FoF1-ATP synthase. The bacterial enzyme also catalyzes ATP hydrolysis. The opposite direction of rotation during ATP synthesis and hydrolysis was confirmed by single-molecule fluorescence resonance energy transfer, FRET, using specific labeling of the rotary subunits γ or e in the F1 motor and the stator subunits [1-3]. The step size in the F1 motor was 120°. In contrast the step size during proton-driven rotation of the c subunits in the Fo motor was 36° using single-molecule FRET. FRET artifacts could be minimized by 'duty cycle optimized alternating laser excitation'. As the two coupled motors of FoF1-ATP synthase showed apparently different step sizes, this mismatch has to be unraveled by mapping the contributions of rotor and stator subunits for transient energy storage. We present the simultaneous observations of F1 and Fo motor rotations using a single-molecule triple FRET approach, which indicate elastic deformations of the rotor between e and c subunits during ATP hydrolysis as well as synthesis.