![]() With this H +:ATP ratio, when 10× the free energy of proton translocation ( \(\triangle \), F 1 overpowers F O and the enzyme hydrolyzes ATP to pump protons. cerevisiae this ratio is 10:3 due to the ten proton-carrying c subunits in F O and three catalytic sites in F 1 8. Even with the rotor turning at hundreds of revolutions per second 5, 6 there is little or no ‘slip’ 7 and the H +:ATP ratio remains constant. Conversely, sequential ATP hydrolysis at each of the three αβ pairs in F 1 causes the γ subunit to turn in the opposite direction, rotating the proton-carrying c-ring against subunit a in F O and pumping protons across the membrane. Rotation of subunit γ within F 1 leads each αβ pairs to cycle through open, tight, and loose conformations that result in the formation of ATP. 1a, green structure), which in yeast is formed from subunits b, d, h, and OSCP (the oligomycin sensitivity conferral protein).ĭuring ATP synthesis, proton translocation through F O at the interface of subunit a and the c-ring causes the γδεc 10 rotor (Fig. Coupling between F 1 and F O requires that the two motors are held stationary relative to each other by a peripheral stalk subcomplex (Fig. In Saccharomyces cerevisiae, the F O region contains subunits a, e, f, g, i/j, k, 8, part of subunit b, and the c 10-ring of the rotor 3, while the F 1 region includes a trimer of catalytic subunit αβ pairs and subunits γ, δ, and ε from the rotor 4. The membrane-embedded F O motor is driven by proton translocation across the membrane through two offset half channels 1, 2 while the soluble F 1 motor is powered by ATP hydrolysis. The enzyme complex consists of two molecular motors positioned to oppose each other’s action on a shared rotor subcomplex (Fig. The structures show how the peripheral stalk opposes the bending force and suggests that during ATP synthesis proton translocation causes accumulation of strain in the stalk, which relaxes by driving the relative rotation of the rotor through six sub-steps within F 1, leading to catalysis.ĪTP synthases use a transmembrane electrochemical proton motive force (pmf) to generate adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganic phosphate (Pi). We used cryoEM to image yeast mitochondrial ATP synthase under strain during ATP-hydrolysis-driven rotary catalysis, revealing a large deformation of the peripheral stalk. Structures of resting mitochondrial ATP synthases revealed a left-handed curvature of the peripheral stalk even though rotation of the rotor, driven by either ATP hydrolysis in F 1 or proton translocation through F O, would apply a right-handed bending force to the stalk. The F 1 and F O motors oppose each other’s action on a shared rotor subcomplex and are held stationary relative to each other by a peripheral stalk. The γ subunit was free to rotate, which could be detected by observing the fluorescence under a fluorescent microscope from the attached actin filament.ATP synthases are macromolecular machines consisting of an ATP-hydrolysis-driven F 1 motor and a proton-translocation-driven F O motor. The whole F1 molecule was fixed to a glass slip through a His-tag such that the a 3β 3 ring was effectively immobilized. Since the γ subunit was too small to visually discern its rotation, Noji et al covalently attached a fluorescein-labeled actin filament to the γ subunit (near where F o would bind). To prove that the γ subunit rotates, you'd have to observe a single molecule. The γ subunit does not appear to undergo any significant conformational change on ATP hydrolysis as evidenced by tritium exchange studies of amide protons. the proton-motive force) causes the γ subunit to rotate like a crankshaft relative to the F1 subunit, forcing the β subunit to change conformation from the T to the O (releasing ATP) and then to the L (binding ADP and Pi) states. The collapse of the proton gradient (i.e. ![]() \): Boyer's three-state conformational model (L-O-T) for ATP synthesis ![]()
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