Torque generation and utilization in the motor enzyme F_oF₁-ATP synthase: half-torque F₁ with short-sized pushrod helix and reduced ATP synthesis by half-torque F_oF₁.

Abstract

ATP synthase (F_oF₁) is made of two motors, a proton-driven motor (F_o) and an ATP-driven motor (F₁), connected by a common rotary shaft, and catalyzes proton flow-driven ATP synthesis and ATP-driven proton pumping. In F₁, the central γ subunit rotates inside the α₃β₃-ring. Here we report structural features of F₁ responsible for torque generation and catalytic ability of the low-torque F_oF₁. (i) Deletion of one or two turns in the α-helix in the C-terminal domain of catalytic β subunit at the rotor/stator contact region generates mutant F₁s, termed F_1(1/2)s, that rotate with about half the normal torque. This helix would support the helix-loop-helix structure acting as a solid ″pushrod″ to push the rotor γ subunit, but the short helix in F_1(1/2)s would fail to accomplish this task. (ii) Three different half-torque F_oF_1(1/2)s were purified and reconstituted into proteoliposomes. They carry out ATP-driven proton-pumping and build up the same small transmembrane ΔpH, indicating that the final ΔpH is directly related to the amount of torque. (iii) The half-torque F_oF_1(1/2)s can catalyze ATP synthesis, though slowly. The rate of synthesis varies widely among the three F_oF_1(1/2)s, which suggests that the rate reflects subtle conformational variations of individual mutants.