With general acceptance of Mitchell’s chemiosmotic mechanism, researchers turned their attention to the structure and operation of ATP synthase. The complex has two principal subcomplexes, F0 and F1, both of which are multimeric proteins (Figure1a). The F0 subcomplex contains three types of integral membrane proteins, designated a, b, and c. In bacteria and in yeast mitochondria, the most common subunit stoichiometry is a1b2c10, but the number of c subunits per sub complex varies among different eukaryotes from 8 to 15. In all cases, the c subunits form a doughnut-shaped ring (“c ring”) in the plane of the membrane. The one a and two b subunits are rigidly linked to one another, but not to the c ring, a critical feature of the protein to which we will return shortly.
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 2. 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 simulta neously 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.
Fig2. 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 12-31. (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 bend ing 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.”]
The F1 subcomplex is a water-soluble complex of five distinct polypeptides with the composition α3β3γδε that is normally firmly bound to the F0 subcomplex at the surface of the membrane. The lower end of the rodlike γ subunit of the F1 subcomplex is a coiled coil that fits into the center of the c-subunit ring of F0 and appears rigidly attached to it. Thus when the c-subunit ring rotates, the rodlike γ subunit moves with it. The F1 ε subunit is rigidly attached to γ and also forms tight contacts with several of the c subunits of F0. The α and β subunits are responsible for the overall globular shape of the F1 subcomplex and associate in alternating order to form a hexamer, αβαβαβ, or (αβ)3, which rests atop the single long γ subunit. The F1 δ subunit is permanently linked to one of the F1 α subunits and also binds to the b subunit of F0. Thus the a and b subunits of the F0 subcomplex and the δ subunit and (αβ)3 hexamer of the F1 subcomplex form a rigid structure anchored in the membrane. The rodlike b subunits form a “stator” that prevents the (αβ)3 hexamer from moving while it rests on the γ subunit, whose rotation, together with that of the c subunits of F0, plays an essential role in the ATP synthesis mechanism described below.
When ATP synthase is embedded in a membrane, the F1 subcomplex forms a knob that protrudes from the cytosolic face (the matrix face in the mitochondrion). Because F1 separated from membranes is capable of catalyzing ATP hydro lysis (ATP conversion to ADP plus Pi) in the absence of the F0 subcomplex, it has been called the F1 ATPase; however, its function in cells is the reverse, to synthesize ATP. ATP hydrolysis is a spontaneous process (ΔG < 0); thus energy is required to drive the ATPase “in reverse” and generate ATP.
