The catalytic domain of the F-ATPase in mitochondria protrudes into the matrix of the organelle, and is attached to the membrane domain by central and peripheral stalks. Energy for the synthesis of ATP from ADP and phosphate is provided by the transmembrane proton-motive-force across the inner membrane, generated by respiration. The proton-motive force is coupled mechanically to ATP synthesis by the rotation at about 100 times per second of the central stalk and an attached ring of c-subunits in the membrane domain. Each c-subunit carries a glutamate exposed around the midpoint of the membrane on the external surface of the ring. The rotation is generated by protonation and deprotonation successively of each glutamate. Each 360°rotation produces three ATP molecules, and requires the translocation of one proton per glutamate by each c-subunit in the ring. In fungi, eubacteria, and plant chloroplasts, ring sizes of c 10

The structures of F1-ATPase from bovine heart mitochondria inhibited with the dietary phytopolyphenol, resveratrol, and with the related polyphenols quercetin and piceatannol have been determined at 2.3-, 2.4-and 2.7-Å resolution, respectively. The inhibitors bind to a common site in the inside surface of an annulus made from loops in the three ␣-and three ␤-subunits beneath the ''crown'' of ␤-strands in their N-terminal domains. This region of F 1-ATPase forms a bearing to allow the rotation of the tip of the ␥-subunit inside the annulus during catalysis. The binding site is a hydrophobic pocket between the C-terminal tip of the ␥-subunit and the ␤TP subunit, and the inhibitors are bound via H-bonds mostly to their hydroxyl moieties mediated by bound water molecules and by hydrophobic interactions. There are no equivalent sites between the ␥-subunit and either the ␤DP or the ␤E subunit. The inhibitors probably prevent both the synthetic and hydrolytic activities of the enzyme by blocking both senses of rotation of the ␥-subunit. The beneficial effects of dietary resveratrol may derive in part by preventing mitochondrial ATP synthesis in tumor cells, thereby inducing apoptosis. mitochondria ͉ oxidative phosphorylation ͉ rotary mechanism ͉ crystal structure

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

The structure of bovine F1-ATPase inhibited by a monomeric form of the inhibitor protein, IF 1, known as I1-60His, lacking most of the dimerization region, has been determined at 2.1-Å resolution. The resolved region of the inhibitor from residues 8 -50 consists of an extended structure from residues 8 -13, followed by two ␣-helices from residues 14 -18 and residues 21-50 linked by a turn. ATP synthase ͉ inhibitor protein ͉ regulation ͉ structure

The structure of bovine F 1 -ATPase, crystallized in the presence of AMP-PNP and ADP, but in the absence of azide, has been determined at 1.9 Å resolution. This structure has been compared with the previously described structure of bovine F 1 -ATPase determined at 1.95 Å resolution with crystals grown under the same conditions but in the presence of azide. The two structures are extremely similar, but they differ in the nucleotides that are bound to the catalytic site in the ␤ DPsubunit. In the present structure, the nucleotide binding sites in the ␤ DP -and ␤ TP -subunits are both occupied by AMP-PNP, whereas in the earlier structure, the ␤ TP site was occupied by AMP-PNP and the ␤ DP site by ADP, where its binding is enhanced by a bound azide ion. Also, the conformation of the side chain of the catalytically important residue, ␣Arg-373 differs in the ␤ DP -and ␤ TP -subunits. Thus, the structure with bound azide represents the ADP inhibited state of the enzyme, and the new structure represents a ground state intermediate in the active catalytic cycle of ATP hydrolysis.Our current understanding of the molecular mechanism of F 1 -ATPase is based on the structural analysis by x-ray crystallography of the enzyme from bovine heart mitochondria. The first high resolution structure (1), now known as the "reference" structure, determined at 2.8 Å resolution with crystals grown in the presence of both ADP and the nonhydrolyzable ATP analog AMP-PNP, 3 showed that the three noncatalytic ␣-subunits and the three catalytic ␤-subunits are arranged in alternation around an asymmetric ␣-helical structure in the single ␥-subunit. The ␣-and ␤-subunits have similar folds consisting of an N-terminal domain with six ␤-strands, a central nucleotide binding domain made of both ␣-helices and ␤-strands and an ␣-helical C-terminal domain containing six ␣-helices in ␤-subunits and seven in ␣-subunits. Because of the asymmetry of the ␥-subunit, the catalytic ␤-subunits adopt different conformations with different nucleotide occupancies. Two of them have similar conformations, but one, designated as ␤ DP , contains bound ADP, and the second, ␤ TP , has bound AMP-PNP. The third has adopted a radically different conformation in which the nucleotide binding domain has been disrupted by an outward hinging movement of part of the domain and the attached C-terminal domain in response to the curvature of the central ␣-helical structure of the ␥-subunit. This ␤-subunit has no bound nucleotide, and so it is known as the "empty" or open state, designated as ␤ E . To explain the interconversion of catalytic sites through "tight," "loose," and "open" states required by a binding change mechanism of catalysis of ATP hydrolysis by F 1 -ATPase (2), it was proposed that the interconversion of sites is effected by a mechanical rotation of the ␥-subunit, each 360°r otation taking each ␤-subunit through the three states and thereby hydrolyzing three ATP molecules. It was shown subsequently that during ATP hydrolysis, either in an ␣ 3 ␤ 3 ␥ complex or in the...

The structure of bovine F 1 -ATPase inhibited with ADP and beryllium fluoride at 2.0 Å resolution contains two ADP.BeF 3 À complexes mimicking ATP, bound in the catalytic sites of the b TP and b DP subunits. Except for a 1 Å shift in the guanidinium of aArg373, the conformations of catalytic side chains are very similar in both sites. However, the ordered water molecule that carries out nucleophilic attack on the c-phosphate of ATP during hydrolysis is 2.6 Å from the beryllium in the b DP subunit and 3.8 Å away in the b TP subunit, strongly indicating that the b DP subunit is the catalytically active conformation. In the structure of F 1 -ATPase with five bound ADP molecules (three in a-subunits, one each in the b TP and b DP subunits), which has also been determined, the conformation of aArg373 suggests that it senses the presence (or absence) of the cphosphate of ATP. Two catalytic schemes are discussed concerning the various structures of bovine F 1 -ATPase.

In mitochondria, the hydrolytic activity of ATP synthase is prevented by an inhibitor protein, IF1. The active bovine protein (84 amino acids) is an alpha-helical dimer with monomers associated via an antiparallel alpha-helical coiled coil composed of residues 49-81. The N-terminal inhibitory sequences in the active dimer bind to two F1-ATPases in the presence of ATP. In the crystal structure of the F1-IF1 complex at 2.8 A resolution, residues 1-37 of IF1 bind in the alpha(DP)-beta(DP) interface of F1-ATPase, and also contact the central gamma subunit. The inhibitor opens the catalytic interface between the alpha(DP) and beta(DP) subunits relative to previous structures. The presence of ATP in the catalytic site of the beta(DP) subunit implies that the inhibited state represents a pre-hydrolysis step on the catalytic pathway of the enzyme.

The structure of the dimeric ATP synthase from bovine mitochondria determined in three rotational states by electron cryo-microscopy provides evidence that the proton uptake from the mitochondrial matrix via the proton inlet half channel proceeds via a Grotthus mechanism, and a similar mechanism may operate in the exit half channel. The structure has given information about the architecture and mechanical constitution and properties of the peripheral stalk, part of the membrane extrinsic region of the stator, and how the action of the peripheral stalk damps the side-to-side rocking motions that occur in the enzyme complex during the catalytic cycle. It also describes wedge structures in the membrane domains of each monomer, where the skeleton of each wedge is provided by three α-helices in the membrane domains of the b-subunit to which the supernumerary subunits e, f, and g and the membrane domain of subunit A6L are bound. Protein voids in the wedge are filled by three specifically bound cardiolipin molecules and two other phospholipids. The external surfaces of the wedges link the monomeric complexes together into the dimeric structures and provide a pivot to allow the monomer–monomer interfaces to change during catalysis and to accommodate other changes not related directly to catalysis in the monomer–monomer interface that occur in mitochondrial cristae. The structure of the bovine dimer also demonstrates that the structures of dimeric ATP synthases in a tetrameric porcine enzyme have been seriously misinterpreted in the membrane domains.