ALTERNATING SITE MECHANISM OF THE FₒF₁ BIOMOLECULAR MOTOR
The FₒF₁ ATP synthase operates as a molecular rotary motor (4). The protein subunits that comprise the rotor and stator of this motor are γ, ε, c and α, β, δ, a, b, b', respectively. The c subunits assemble into a ring that is currently estimated to contain 14 subunits. Each transmembrane helical c subunit has an aspartic acid that is protonated by the protons of the lumen via a channel located on subunit a. An arginine at a separate location on subunit a displaces the proton from subunit c to release it to the stroma. Each successive proton displacement induces the stepwise rotation of the c subunit ring, driven by the transmembrane proton gradient.
The γ and ε subunits, docked to the c subunit ring, also rotate in response to the proton gradient-driven step-wise rotation and comprise the rotor of the biomolecular motor. The N and C termini of the γ subunit form a coiled-coil that protrudes through a ring composed of three α and β subunit heterodimers that comprise each catalytic site. The ring of αβ heterodimers along with subunits a, b, b', and δ comprise the stator of the motor.
Each catalytic site adopts a conformation in response to the rotational position of the γ subunit such that the three catalytic sites are in different conformations with one site empty. Completion of a catalytic cycle at any one site requires 360° rotation of the γ subunit. In what is known as the binding-change or alternating site mechanism, the binding of a Mg²⁺-ADP complex and Pi to the empty site triggers a 120° step rotation of the γ subunit, driven by the proton gradient, that releases ATP from a different catalytic site (5). The energy from the flux of 3 protons through the membrane is minimally required to drive a 120° rotation and ATP production, although the measured ratio of protons/ATP is ~ 4.
The FₒF₁ ATP synthase uses the nonequilibrium proton gradient to drive the reaction, ADP + Pi = ATP + H₂O, far beyond the point of equilibrium, in favor of the products. Most enzymes that use ATP hydrolysis as a source of energy, derive the energy from the reaction by returning the concentration ratio of ATP/ADP+Pi toward equilibrium. Sequential changes in catalytic site conformation maintain this ratio away from equilibrium. In the initial conformation, the binding of ADP and Pi is preferred to ATP. The first conformational change increases the affinity of the catalytic site for substrates and products, and has a low activation energy barrier for ATP synthesis that allows rapid interconversion of substrates and products. The second conformational change converts the site to one that favors ATP over ADP and Pi, while the third decreases the affinity for ATP so that, even when the ATP/ADP+Pi ratio is high, ATP hydrolysis is minimized and ATP dissociates.