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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[degrees] 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.sub.10]-[c.sub.15] subunits have been observed, implying that these enzymes need 3.3-5 protons to make each ATP, but until now no higher eukaryote has been examined. As shown here in the structure of the bovine [F.sub.1]-c-ring complex, the c-ring has eight c-subunits. As the sequences of c-subunits are identical throughout almost all vertebrates and are highly conserved in invertebrates, their F-ATPases probably contain [c.sub.8]-rings also. Therefore, in about 50,000 vertebrate species, and probably in many or all of the two million invertebrate species, 2.7 protons are required by the F-ATPase to make each ATP molecule. ATP synthase | rotational catalysis | c-ring structure | protons per ATP | vertebrates doi/ 10.1073/pnas.1011099107

The structure of bovine [F.sub.1]-ATPase inhibited by a monomeric form of the inhibitor protein, I[F.sub.1], known as I1-60His, lacking most of the dimerization region, has been determined at 2.1-[Angstrom] resolution. The resolved region of the inhibitor from residues 8-50 consists of an extended structure from residues 8-13, followed by two [alpha]-helices from residues 14-18 and residues 21-50 linked by a turn. The binding site in the [[beta].sub.DP]-[[alpha].sub.DP] catalytic interface is complex with contributions from five different subunits of [F.sub.1]-ATPase. The longer helix extends from the external surface of [F.sub.1] via a deep groove made from helices and loops in the C-terminal domains of subunits [[beta].sub.DP], [[alpha].sub.DP], [[beta].sub.TP], and [[alpha].sub.TP] to the internal cavity surrounding the central stalk. The linker and shorter helix interact with the [gamma]-subunit in the central stalk, and the N-terminal region extends across the central cavity to interact with the nucleotide binding domain of the [[alpha].sub.E] subunit. To form these complex interactions and penetrate into the core of the enzyme, it is likely that the initial interaction of the inhibitor with [F.sub.1] forms via the open conformation of the [[beta].sub.E] subunit. Then, as two ATP molecules are hydrolyzed, the [[beta].sub.E]-[[alpha].sub.E] interface converts to the [[beta].sub.DP]-[[alpha].sub.DP] interface via the [[beta].sub.TP]-[[alpha].sub.TP] interface, trapping the inhibitor progressively in its binding site and a nucleotide in the catalytic site of subunit [[beta].sub.DP]. The inhibition probably arises by I[F.sub.1] imposing the structure and properties of the [[beta].sub.TP]-[[alpha].sub.TP] interface on the [[beta].sub.DP]-[[alpha].sub.DP] interface, thereby preventing it from hydrolyzing the bound ATP. ATP synthase | inhibitor protein | regulation | structure

The structures of [F.sub.1]-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-[Angstrom] resolution, respectively. The inhibitors bind to a common site in the inside surface of an annulus made from loops in the three [alpha]- and three [beta]-subunits beneath the 'crown' of [beta]-strands in their N-terminal domains. This region of [F.sub.1]-ATPase forms a bearing to allow the rotation of the tip of the [gamma]-subunit inside the annulus during catalysis. The binding site is a hydrophobic pocket between the C-terminal tip of the [gamma]-subunit and the [[beta].sub.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 [gamma]-subunit and either the [[beta].sub.DP] or the [[beta].sub.E] subunit. The inhibitors probably prevent both the synthetic and hydrolytic activities of the enzyme by blocking both senses of rotation of the [gamma]-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

In the structure of bovine [F.sub.1]-ATPase determined at 1.95-[Angstrom] resolution with crystals grown in the presence of ADP, 5'-adenylylimidodiphosphate, and azide, the azide anion interacts with the [beta]-phosphate of ADP and with residues in the ADP-binding catalytic subunit, [[beta].sub.DP]. It occupies a position between the catalytically essential amino acids, [beta]-Lys-162 in the P loop and the 'arginine finger' residue, [alpha]-Arg-373, similar to the site occupied by the [gamma],-phosphate in the ATP-binding subunit, [[beta].sub.TP]. Its presence in the [[beta].sub.DP]-subunit tightens the binding of the side chains to the nucleotide, enhancing its affinity and thereby stabilizing the state with bound ADP. This mechanism of inhibition appears to be common to many other ATPases, including ABC transporters, SecA, and DNA topoisomerase II[alpha]. It also explains the stimulatory effect of azide on ATP-sensitive potassium channels by enhancing the binding of ADP. mitochondria | oxidative phosphorylation | inhibition | mechanism

The bovine inhibitor subunit was expressed in Escherichia coli and purified in high yield to gather data on the mechanism of the mitochondrial ATPase inhibitor subunit. The minimal inhibitory sequence of the inhibitor subunit was found to consist of the residues 14-47. The immediately adjacent sequences 10-13 and 48-56 help to stabilize the complex between F1F0-ATPase and the inhibitor protein, and residues 1-9 adn 57-84 seem to be unessential.