The Role of Pi-Release as the Main Torque Generating Step of F1-Atpase

Abstract

F1-ATPase (α3β3γ) is a rotary motor protein, which couples ATP hydrolysis to the rotary motion. Extensive studies on F1-ATPase revealed that each of three β-subunits, which has the catalytic site, follows the same reaction pathway of ATP hydrolysis, but they are always in a reaction phase differing by ±120° from each other. When we focus on one β-subunit, the β binds ATP at a particular binding angle. After the γ rotates 200°, the β cleavages the bound ATP into ADP and Pi. The produced ADP and Pi are released from the β after further 40°and 120o rotation, at +240° and +320o from the ATP-binding angle, respectively. In this study, we observed the rotating F1 and measured the equilibrium of ATP cleavage and synthesis at the single molecule level. As F1 released the produced Pi, the equilibrium was shifted to ATP cleavage; therefore, from the time course of the probability of ATP cleavage, we determined the rate of Pi-release at the angles for ATP cleavage and ADP release as 0.021s−1and 0.94 s−1, respectively. We also determined the rate at the proper angle for Pi-release as 2,600 s−1 by using the fast-framing camera with 18,000 fps. From these results, we found that the rate of Pi release strongly depended on the rotary angle, and the dependence of activation energy on the rotary angle was determined to be ΔE=5.5 kBT/rad, which was almost 55% of the net rotary torque of F1.