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Adenosine triphosphate (ATP), the chemical energy currency of biology, is synthesized in eukaryotic cells primarily by the mitochondrial ATP synthase. ATP synthases operate by a rotary catalytic mechanism where proton translocation through the membrane-inserted FO region is coupled to ATP synthesis in the catalytic F1 region via rotation of a central rotor subcomplex. We report here single particle electron cryomicroscopy (cryo-EM) analysis of the bovine mitochondrial ATP synthase. Combining cryo-EM data with bioinformatic analysis allowed us to determine the fold of the a subunit, suggesting a proton translocation path through the FO region that involves both the a and b subunits. 3D classification of images revealed seven distinct states of the enzyme that show different modes of bending and twisting in the intact ATP synthase. Rotational fluctuations of the c8-ring within the FO region support a Brownian ratchet mechanism for proton-translocation-driven rotation in ATP synthases. DOI: http://dx.doi.org/10.7554/eLife.10180.001, eLife digest A molecule called adenosine triphosphate (ATP) is the energy currency in cells. Most of the ATP used by cells is made by the membrane-embedded enzyme ATP synthase. This enzyme is found in membranes inside specialized compartments known as mitochondria. ATP synthase is made up of many protein subunits that work together as a molecular machine. Hydrogen ions flow across the membrane through the ATP synthase, turning a rotor structure within the enzyme, which leads to the production of ATP. It is not known how the transport of hydrogen ions causes rotation of the rotor. Some researchers have proposed that the enzyme works as a ratchet that is driven by the random Brownian motion of the rotor. That is, the rotational position of the rotor fluctuates randomly, but a ratchet mechanism ensures that there is a net rotation in one direction. However, there is currently little experimental evidence to back up this theory, which is known as the Brownian ratchet model. Zhou, Rohou et al. used a technique called electron cryomicroscopy (or cryo-EM) to study ATP synthase from cows. The cryo-EM data made it possible to use computer software to construct a three-dimensional model of the enzyme that is more detailed than previous attempts. Zhou, Rohou et al. show that the structure of ATP synthase is flexible, with the different protein subunits bending, flexing, and rotating relative to each other. This variability in the position of the rotor is consistent with the Brownian ratchet model. Together, these findings reveal important new details about the structure of ATP synthase and provide some of the first experimental evidence for the Brownian ratchet model. The new three-dimensional structure of ATP synthase will open the door to testing hypotheses of how the ATP synthase works. DOI: http://dx.doi.org/10.7554/eLife.10180.002

The rotation of the central stalk of F 1 -ATPase is driven by energy derived from the sequential binding of an ATP molecule to its three catalytic sites and the release of the products of hydrolysis. In human F 1 -ATPase, each 360° rotation consists of three 120° steps composed of substeps of about 65°, 25°, and 30°, with intervening ATP binding, phosphate release, and catalytic dwells, respectively. The F 1 -ATPase inhibitor protein, IF 1 , halts the rotary cycle at the catalytic dwell. The human and bovine enzymes are essentially identical, and the structure of bovine F 1 -ATPase inhibited by IF 1 represents the catalytic dwell state. Another structure, described here, of bovine F 1 -ATPase inhibited by an ATP analog and the phosphate analog, thiophosphate, represents the phosphate binding dwell. Thiophosphate is bound to a site in the α E β E -catalytic interface, whereas in F 1 -ATPase inhibited with IF 1 , the equivalent site is changed subtly and the enzyme is incapable of binding thiophosphate. These two structures provide a molecular mechanism of how phosphate release generates a rotary substep as follows. In the active enzyme, phosphate release from the β E -subunit is accompanied by a rearrangement of the structure of its binding site that prevents released phosphate from rebinding. The associated extrusion of a loop in the β E -subunit disrupts interactions in the α E β E -catalytic interface and opens it to its fullest extent. Other rearrangements disrupt interactions between the γ-subunit and the C-terminal domain of the α E -subunit. To restore most of these interactions, and to make compensatory new ones, the γ-subunit rotates through 25°–30°.

The structures of F-ATPases have been determined predominantly with mitochondrial enzymes, but hitherto no F-ATPase has been crystallized intact. A high-resolution model of the bovine enzyme built up from separate sub-structures determined by X-ray crystallography contains about 85% of the entire complex, but it lacks a crucial region that provides a transmembrane proton pathway involved in the generation of the rotary mechanism that drives the synthesis of ATP. Here the isolation, characterization and crystallization of an integral F-ATPase complex from the α-proteobacterium Paracoccus denitrificans are described. Unlike many eubacterial F-ATPases, which can both synthesize and hydrolyse ATP, the P. denitrificans enzyme can only carry out the synthetic reaction. The mechanism of inhibition of its ATP hydrolytic activity involves a ζ inhibitor protein, which binds to the catalytic F₁-domain of the enzyme. The complex that has been crystallized, and the crystals themselves, contain the nine core proteins of the complete F-ATPase complex plus the ζ inhibitor protein. The formation of crystals depends upon the presence of bound bacterial cardiolipin and phospholipid molecules; when they were removed, the complex failed to crystallize. The experiments open the way to an atomic structure of an F-ATPase complex., his work was funded by the intramural programme of the Medical Research Council via MRC programme U105663150 to J.E.W., and by support from the Biotechnology and Biological Sciences Research Council to M.J.O.W.

The mitochondrial F 1 -ATPase inhibitor protein, IF 1 , inhibits the hydrolytic, but not the synthetic activity of the F-ATP synthase, and requires the hydrolysis of ATP to form the inhibited complex. In this complex, the α-helical inhibitory region of the bound IF 1 occupies a deep cleft in one of the three catalytic interfaces of the enzyme. Its N-terminal region penetrates into the central aqueous cavity of the enzyme and interacts with the γ-subunit in the enzyme's rotor. The intricacy of forming this complex and the binding mode of the inhibitor endow IF 1 with high specificity. This property has been exploited in the development of a highly selective affinity procedure for purifying the intact F-ATP synthase complex from mitochondria in a single chromatographic step by using inhibitor proteins with a C-terminal affinity tag. The inhibited complex was recovered with residues 1–60 of bovine IF 1 with a C-terminal green fluorescent protein followed by a His-tag, and the active enzyme with the same inhibitor with a C-terminal glutathione- S -transferase domain. The wide applicability of the procedure has been demonstrated by purifying the enzyme complex from bovine, ovine, porcine and yeast mitochondria. The subunit compositions of these complexes have been characterized. The catalytic properties of the bovine enzyme have been studied in detail. Its hydrolytic activity is sensitive to inhibition by oligomycin, and the enzyme is capable of synthesizing ATP in vesicles in which the proton-motive force is generated from light by bacteriorhodopsin. The coupled enzyme has been compared by limited trypsinolysis with uncoupled enzyme prepared by affinity chromatography. In the uncoupled enzyme, subunits of the enzyme's stator are degraded more rapidly than in the coupled enzyme, indicating that uncoupling involves significant structural changes in the stator region.