Torque generation and utilization in motor enzyme F ₀F ₁-ATP synthase: Half-torque F ₁ with short-sized pushrod helix and reduced ATP synthesis by half-torque F ₀F ₁

ATP synthase (F ₀F ₁) is made of two motors, a proton-driven motor (F ₀) and an ATP-driven motor (F ₁), connected by a common rotary shaft, and catalyzes proton flow-driven ATP synthesis and ATP-driven proton pumping. In F ₁, the central γ subunit rotates inside the α ₃β ₃ ring. Here we report structural features of F ₁ responsible for torque generation and the catalytic ability of the low-torque F ₀F ₁. (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 F ₁s, termed F ₁(1/2)s, that rotate with about half of 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 ₀F 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 ₀F ₁(1/2)s can catalyzeATPsynthesis, although slowly. The rate of synthesis varies widely among the three F ₀F ₁(1/2)s, which suggests that the rate reflects subtle conformational variations of individual mutants.