Each copy of the c subunit contains two membrane-spanning α helices that form a hairpin-like structure. An aspartate residue, Asp-61 (E. coli ATPase numbering), in the center of one of these helices in each c subunit is thought to play a key role in proton movement by binding and releasing protons as they traverse the membrane. Chemical modification of this aspartate by the poison dicyclohexylcarbodiimide, or its mutation to alanine, specifically blocks proton movement through F0. According to one current model, the protons traverse the membrane via two staggered half-channels, I and II (see Figure 1a and b). They are called half-channels because each ex tends only halfway across the membrane; the intramembrane termini of the channels are at the level of Asp-61 in the middle of the membrane. Half-channel I is open only to the exoplasmic face, and half-channel II is open only to the cytosolic face. Prior to rotation, each of the Asp-61 carboxylate side chains in the c subunits is bound to a proton, except that on the c subunit in contact with half-channel I. The negative charge on that unprotonated carboxylate (the “empty” proton-binding site; see Figure 1b, bottom) is neutralized by interaction with the positively charged side chain of Arg-210 from the a subunit. Proton translocation across the membrane begins when a proton from the exoplasmic medium moves upward through half-channel I (Figure 1b, step 1). As that pro ton moves into the empty proton-binding site, it displaces the Arg-210 side chain, which swings toward the filled proton binding site of the adjacent c subunit in contact with half channel II (step 2). As a consequence, the positive side chain of Arg-210 displaces the proton bound to Asp-61 of the ad jacent c subunit. This displaced proton is now free to travel up half-channel II and out into the cytosolic medium (step 3). Thus when one proton entering from half-channel I binds to the c ring, a different proton is released to the opposite side of the membrane via half-channel II. Rotation of the entire c ring due to thermal/Brownian motion (step 4) then allows the newly unprotonated c subunit to move into alignment above half-channel I as an adjacent, protonated c subunit rotates in to take its place under half-channel II. The entire cycle is then repeated (step 5) as additional protons move down their electrochemical gradient from the exoplasmic medium to the cytosolic medium. During each partial rotation (360° divided by the number of c subunits in the ring), the c ring rotation is ratcheted, in that net movement of the ring occurs in only one direction. The energy driving the protons across the mem brane, and thus the rotation of the c ring, comes from the electrochemical gradient across the membrane. If the direction of proton flow is reversed, which can be done by experimentally reversing the direction of the proton gradient and the proton motive force, the direction of c ring rotation is reversed.
Fig1. Structure of ATP synthase (the F0F1 complex) in the bacterial plasma membrane and mechanism of proton translocation across the membrane. (a) The F0 membrane-embedded subcomplex of ATP synthase is built of three integral membrane proteins: one copy of a, two copies of b, and an average of ten copies of c arranged in a ring in the plane of the membrane. Two proton half-channels near the interfaces of subunit a with the c subunits mediate proton movement across the membrane (proton path is indicated by red arrows). Half-channel I allows protons to move one at a time from the exoplasmic medium (equivalent to intermembrane space in mitochondria) to the negatively charged side chain of Asp-61 in the center of a c subunit near the middle of the membrane. The proton-binding site in each c subunit is represented as a white circle with a blue “−” representing the negative charge on the side chain of Asp-61. Half-channel II permits protons to move from the Asp-61 of an adjacent c subunit into the cytosolic medium. The detailed structure of the c ring and a portion of the adjacent a subunit is shown in Figure 3. The F1 subcomplex of ATP synthase contains three copies each of subunits α and β, which form a hexamer resting atop the single rod-shaped γ subunit, which is inserted into the c ring of F0. The ε subunit is rigidly attached to the γ subunit and also to several of the c subunits. The δ subunit permanently links one of the α subunits in the F1 subcomplex to the b subunit of F0. Thus the F0 a and b sub units and the F1 δ subunit and (αβ)3 hexamer form a rigid structure (orange) anchored in the membrane. During proton flow, the c ring and the attached F1 ε and γ subunits rotate as a unit (green), causing conformational changes in the F1 β subunits, leading to ATP synthesis. (b) Potential mechanism of proton translocation. Step 1 : A proton from the exoplasmic space enters half-channel I and moves toward the “empty” (unprotonated) Asp-61 proton-binding site. The negative charge (blue “−”) on the unprotonated side chain Asp-61 is balanced, in part, by a positive charge on the side chain of Arg-210 (red “+”). Step 2: The proton fills the empty proton-binding site and simultaneously displaces the positively charged Arg-210 side chain, which swings over to the filled proton-binding site on the adjacent c subunit (curved arrow). As a consequence, the proton bound at that adjacent site is displaced. Step 3 : The displaced adjacent proton moves through half-channel II and is released into the cytosolic space, leaving an empty proton-binding site on Asp-61. Step 4: Counterclockwise rotation of the entire c ring moves the “empty” c subunit over half-channel I. Step 5: The process is repeated. See M. J. Schnitzer, 2001, Nature 410:878; P. D. Boyer, 1999, Nature 402:247; and C. von Ballmoos, A. Wiedenmann, and P. Dimroth, 2009, Annu. Rev. Biochem. 78:649.
Because the γ subunit of F1 is tightly attached to the c ring of F0, rotation of the c ring associated with proton movement causes rotation of the γ subunit. According to the binding change mechanism, a 120° rotation of γ powers synthesis of one ATP (see Figure 2). Thus complete rotation of the c ring by 360° would generate three ATPs. In E. coli, where the F0 composition is a1b2c10, movement of 10 protons drives one complete rotation and thus synthesis of three ATPs. This value is consistent with experimental data on proton flux during ATP synthesis, providing indirect support for the model coupling proton movement to c ring rotation depicted in Figure 1. The F0 from chloroplasts contains 14 c subunits per ring, and movement of 14 protons would be needed for synthesis of three ATPs. Why these otherwise similar F0F1 complexes have evolved to have different H+:ATP ratios is not clear.
Fig2. The binding-change mechanism of ATP synthesis from ADP and Pi. This view is looking up at F1 from the membrane sur face (see Figure 1). As the γ subunit rotates by 120° in the center, each of the otherwise identical F1 β subunits alternates between three conformational states (O, open, with oval representation of the binding site; L, loose, with a rectangular binding site; T, tight, with a triangular site) that differ in their binding affinities for ATP, ADP, and Pi. The cycle begins (upper left) when ADP and Pi bind loosely to one of the three β subunits (here, arbitrarily designated β1) whose nucleotide-binding site is in the O (open) conformation. Proton flux through the F0 portion of the protein powers a 120° rotation of the γ subunit (relative to the fixed β subunits) (step 1). This causes the rotating γ subunit, which is asymmetric, to push differentially against the β subunits, resulting in a conformational change and an increase in the binding affinity of the β1 subunit for ADP and Pi (O → L), an increase in the binding affinity of the β3 subunit for ADP and Pi that were previously bound (L → T), and a decrease in the binding affinity of the β2 subunit for a previously bound ATP (T → O), causing release of the bound ATP. Step 2: Without additional rotation, the ADP and Pi in the T site (here, in the β3 subunit) form ATP, a reaction that does not require an input of additional energy due to the special environment in the active site of the T state. At the same time, a new ADP and Pi bind loosely to the unoccupied O site on β2. Step 3: Proton flux powers another 120° rotation of the γ sub unit, consequent conformational changes in the binding sites (L → T, O → L, T → O), and release of ATP from β3. Step 4: Without additional rotation, the ADP and Pi in the T site of β1 form ATP, and additional ADP and Pi bind to the unoccupied O site on β3. The process continues with rotation (step 5) and ATP formation (step 6) until the cycle is complete, with three ATPs having been produced for every 360° rotation of γ. See P. Boyer, 1989, FASEB J. 3:2164; Y. Zhou et al., 1997, Proc. Natl. Acad. Sci. USA 94:10583; and M. Yoshida, E. Muneyuki, and T. Hisabori, 2001, Nat. Rev. Mol. Cell Biol. 2:669.
High-resolution electron microscopic tomography (Figure 3) has provided additional insights into the structure of the c ring/a subunit interface and other features of F0F1 structure and function. The experiments were performed using F0F1 either dissolved in detergent, then incorporated into artificial phospholipid bilayers, or in isolated mitochondrial membranes. Figures 3a and b show two views of the two membrane-spanning α helices in each copy of the c subunit (green) that together form the c ring. In a portion of the a subunit (orange), a bundle of four α helices that are almost parallel to and embedded within the inner mitochondrial membrane forms the interface with the c ring and positions the side chain of Arg 210 adjacent to the c ring so that it can mediate proton displacement from Asp 61 as shown in Figure 1. The c ring/a subunit interface also forms the two proton half-channels through which protons flow out of the intermembrane space (red arrow), around the c ring (black arrows in Figure 3b), and then out into the ma trix (red arrow). Each F0F1 monomer bends the membrane by approximately 43° (Figure 3c). The monomers dimerize to impart high membrane curvature (~86°) and then align in long rows, contributing to the formation of the edges and tips of the pancake-like (flat) and tubular cristae (Figure 3d).
Fig3. High-resolution electron microscopy-based mechanism of proton translocation and bending of cristae membranes by ATP synthase. (a) and (b) The interface be tween the c ring (green) and a subunit (orange) of detergent-solubilized mitochondrial ATP synthase from the alga Polytomella sp., imaged by single-particle cryoelectron microscopy (∼0.62 nm resolution), is shown (a) from within the plane of the inner mitochondrial membrane (side view) and (b) after a 90° rotation (top view). The movement of protons through half-channels I and II and the rotation of the c ring are described in detail in Figure 1. (a) Cross section through the c ring (right) shows that each c subunit is a transmembrane helical hairpin – two adjacent transmembrane α helices connected by a short nonhelical linker on the matrix side of the membrane. The negative side chain of the c subunit’s Asp61 in the middle of the membrane is thought to both serve as a binding site for translocating protons and interact with the side chain of the a subunit’s Arg210. (c) A model of the bovine heart mitochondrial ATP synthase is based on cryoelectron tomography and electron crystallographic image processing from crystalline ATP synthase in artificial membranes. Each F0F1 monomer bends the membrane by ∼43° toward the intermembrane space (IMS), resulting in dimers bending the membrane by ~86°. The rotating c ring and γ and ε subinits are colored green, and the remaining static portions of the enzyme are shown in orange. (d) Cryoelectron tomographic image of frozen membranes from purified Saccharomyces cerevisiae (yeast) mitochondria. The surfaces of the ATP synthase complexes (orange) and the membrane (gray) show that the enzymes dimerize as in (c) and align into long rows that bend the membranes into characteristic tubular and flat, pancake like cristae. [Parts (a) and (b) reprinted by permission from Macmillan Publishers Ltd., from Allegretti, M., et al., “Horizontal membrane-intrinsic α-helices in the stator a-subunit of an F-type ATP synthase,” Nature, 2015, 521, pp 237-240, 2015; permission conveyed through the Copyright Clearance Center, Inc. Part (c) data from C. Jiko et al., 2015, eLife 4:e06119. Part (d) from Proc. Natl. Acad. Sci. USA 2012. 109(34):13602-13607, Fig. 4C and D. “Structure of the yeast F1Fo-ATP synthase dimer and its role in shaping the mitochondrial cristae.”]
