Molecular switch of F0F1-ATP synthase,G-protein,and other ATP-driven enzymes

Exchange-out of amide tritium from labeled -subunit of ₃₃ complex of F₀F₁-ATP synthase was not accelerated by ATP, suggesting that hemagglutinin-type transition of coiled-coil structure did not occur in -subunit. Local topology of nucleotide binding site and switch II region of G-protein resemble those of F₁- subunit and other proteins which catalyze ATP-triggered reactions. Probably, binding of nucleotide to F₀F₁-ATP synthase induces conformational change of the switch II-like region with transforming subunit structure from open to closed form and this transformation results in loss of hydrogen bonds with the subunit, thus enabling the subunit to move.     

Modulation at a Distance of Proton Conductance through the Saccharomyces cerevisiae Mitochondrial F1F0-ATP Synthase by Variants of the Oligomycin Sensitivity-Conferring Protein Containing Substitutions near the C-Terminus

We have sought to elucidate how the oligomycin sensitivity-conferring protein (OSCP) of the mitochondrial F₁F₀-ATP synthase (mtATPase) can influence proton channel function. Variants of OSCP, from the yeast Saccharomyces cerevisiae, having amino acid substitutions at a strictly conserved residue (Gly166) were expressed in place of normal OSCP. Cells expressing the OSCP variants were able to grow on nonfermentable substrates, albeit with some increase in generation time. Moreover, these strains exhibited increased sensitivity to oligomycin, suggestive of modification in functional interactions between the F₁ and F₀ sectors mediated by OSCP. Bioenergetic analysis of mitochondria from cells expressing OSCP variants indicated an increased respiratory rate under conditions of no net ATP synthesis. Using specific inhibitors of mtATPase, in conjunction with measurement of changes in mitochondrial transmembrane potential, it was revealed that this increased respiratory rate was a result of increased proton flux through the F₀ sector. This proton conductance, which is not coupled to phosphorylation, is exquisitely sensitive to inhibition by oligomycin. Nevertheless, the oxidative phosphorylation capacity of these mitochondria from cells expressing OSCP variants was no different to that of the control. These results suggest that the incorporation of OSCP variants into functional ATP synthase complexes can display effects in the control of proton flux through the F₀ sector, most likely mediated through altered protein—protein contacts within the enzyme complex. This conclusion is supported by data indicating impaired stability of solubilized mtATPase complexes that is not, however, reflected in the assembly of functional enzyme complexes in vivo. Given a location for OSCP atop the F₁-₃₃ hexamer that is distant from the proton channel, then the modulation of proton flux by OSCP must occur at a distance. We consider how subtle conformational changes in OSCP may be transmitted to F₀.     

Evolutionary relationship between Enterobacteriaceae: comparison of the ATP synthases (F1F0) of Escherichia coli and Klebsiella pneumoniae

The ATP synthase complex of Klebsiella pneumoniae (KF₁F₀) has been purified and characterized. SDS-gel electrophoresis of the purified F₁F₀ complexes revealed an identical subunit pattern for E. coli (EF₁F₀) and K. pneumoniae. Antibodies raised against EF₁ complex and purified EF₀ subunits recognized the corresponding polypeptides of EF₁F₀ and KF₁F₀ in immunoblot analysis. Protease digestion of the individual subunits generated an identical cleavage pattern for subunits , , , , a, and c of both enzymes. Only for subunit different cleavage products were obtained. The isolated subunit c of both organisms showed only a slight deviation in the amino acid composition. These data suggest that extensive homologies exist in primary and secondary structure of both ATP synthase complexes reflecting a close phylogenetic relationship between the two enterobacteric tribes.Abbreviations ACMA 9-amino-6-chloro-2-methoxyacridine - DCCD N,N-dicyclohexylcarbodiimide - FITC fluorescein isothiocyanate - SDS sodium dodecyl sulfate - TTFB 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole     

Evidence from immunological studies of structure-mechanism relationship of F1 and F1F0

Monoclonal and polyclonal antibodies directed against peptides of F₁-ATPase or F₁F₀-ATPase synthase provide new and efficient tools to study structure-function relationships and mechanisms of such complex membrane enzymes. This review summarizes the main results obtained using this approach. Antibodies have permitted the determination of the nature of subunits involved in the complex, their stoichiometry, their organization, neighboring interactions, and vectorial distribution within or on either face of the membrane. Moreover, in a few cases, amino acid sequences exposed on a face of the membrane or buried inside the complex have been identified. Antibodies are very useful for detecting the role of each subunit, especially for those subunits which appear to have no direct involvement in the catalytic mechanism. Concerning the mechanisms, the availability of monoclonal antibodies which inhibit (or activate) ATP hydrolysis or ATP synthesis, which modify nucleotide binding or regulation of activities, which detect specific conformations, etc. brings many new ways of understanding the precise functions. The specific recognition by monoclonal antibodies on the subunit of epitopes in the proximity of, or in the catalytic site, gives information on this site. The use of anti- monoclonal antibodies has shown asymmetry of in the complex as already shown for . In addition, the involvement of with respect to nucleotide site cooperativity has been detected. Finally, the formation of F₁F₀-antibody complexes of various masses, seems to exclude the functional rotation of F₁ around F₀ during catalysis.Abbreviations IF₁ natural protein inhibitor of the ATPase-ATP synthase - OSCP oligomycin sensitivity-conferring protein - DCCD dicyclohexylcarbodiimide - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoreses - F₁ F₁-ATPase, coupling factor F₁ of ATPase - F₁F₀ F₁F₀-ATP synthase, ATPase-ATP synthase complex     

The Structural and Functional Connection between the Catalytic and Proton Translocating Sectors of the Mitochondrial F 1 F 0 -ATP Synthase

The structural and functional connection between the peripheral catalytic F₁ sector and theproton-translocating membrane sector F₀ of the mitochondrial ATP synthase is reviewed. Theobservations examined show that the N-terminus of subunit , the carboxy-terminal and centralregion of F₀I-PVP(b), OSCP, and part of subunit d constitute a continuous structure, the lateralstalk, which connects the peripheries of F₁ to F₀ and surrounds the central element of thestalk, constituted by subunits and . The ATPase inhibitor protein (IF₁) binds at one sideof the F₁F₀ connection. The carboxy-terminal segment of IF₁ apparently binds to OSCP. The42L-58K segment of IF₁, which is per se the most active domain of the protein, binds at thesurface of one of the three / pairs of F₁, thus preventing the cyclic interconversion of thecatalytic sites required for ATP hydrolysis.     

The native F0F1-inhibitor protein complex from beef heart mitochondria and its reconstitution in liposomes

A functional F₀F₁ ATP synthase that contains the endogenous inhibitor protein (F₀F₁I) was isolated by the use of two combined techniques [Adolfsen, R., McClung, J.A., and Moudrianakis, E. N. (1975).Biochemistry 14, 1727–1735; Dreyfus, G., Celis, H., and Ramirez, J. (1984).Anal. Biochem. 142, 215–220]. The preparation is composed of 18 subunits as judged by SDS-PAGE. A steady-state kinetic analysis of the latent ATP synthase complex at various concentrations of ATP showed aV _max of 1.28mol min^–1 mg^–1, whereas theV _max of the complex without the inhibitor was 8.3mol min^–1 mg^–1. In contrast, theK _m for Mg-ATP of F₀F₁ I was 148M, comparable to theK _m value of 142M of the F₀F₁ complex devoid of IF₁. The hydrolytic activity of the F₀F₁I increased severalfold by incubation at 60C at pH 6.8, reaching a maximal ATPase activity of 9.5mol min^–1 mg^–1; at pH 9.0 a rapid increase in the specific activity of hydrolysis was followed by a sharp drop in activity. The latent ATP synthase was reconstituted into liposomes by means of a column filtration method. The proteoliposomes showed ATP-Pi exchange activity which responded to phosphate concentration and was sensitive to energy transfer inhibitors like oligomycin and the uncouplerp-trifluoromethoxyphenylhydrazone.     

The photosynthetic F1-α3β3 and α1β1 catalytic core complexes

Minimal photosynthetic catalytic F₁() core complexes, containing equimolar ratios of the and subunits, were isolated from membrane-bound spinach chloroplast CF₁ and Rhodospirillum rubrum chromatophore RrF₁. A CF₁-₃₃ hexamer and RrF₁-₁₁ dimer, which were purified from the respective F₁() complexes, exhibit lower rates and different properties from their parent F₁-ATPases. Most interesting is their complete resistance to inhibition by the general F₁ inhibitor azide and the specific CF₁ inhibitor tentoxin. These inhibitors were earlier reported to inhibit multisite, but not unisite, catalysis in all sensitive F₁-ATPases and were therefore suggested to block catalytic site cooperativity. The absence of this typical property of all F₁-ATPases in the ₁₁ dimer is consistant with the view that the dimer contains only a single catalytic site. The ₃₃ hexamer contains however all F₁ catalytic sites. Therefore the observation that CF₁-₃₃ can bind tentoxin and is stimulated by it suggests that the F₁ subunit, which is required for obtaining inhibition by tentoxin as well as azide, plays an important role in the cooperative interactions between the F₁-catalytic sites.Abbreviations CF₀F₁ chloroplast F₀F₁ - CF₁ chloroplast F₁ - CF₁ chloroplast F₁ subunit - CF₁ chloroplast F₁ subunit - CF₁() a complex containing equal amounts of the CF₁ and subunits - MF₁ mitochondrial F₁ - RrF₀F₁ Rhodospirillum rubrum F₀F₁ - RrF₁ R. rubrum F₁ - RrF₁ R. rubrum F₁ subunit - RrF₁ R. rubrum F₁ subunit - RrF₁() a complex containing equal amounts of the RrF₁ and subunits - Rubisco Ribulose-1,5-bisphosphate carboxylase - TF₁ thermophilic bacterium PS3 F₁     

Structure of F1-ATPases

F₁-ATPases are large multimeric proteins that can be isolated from the membrane bound system that catalyzes the phosphorylation of ADP by inorganic phosphate in bacteria, plants, and mitochondria. They can be visualized in electron micrographs of the inner mitochondrial membranes where they appear as large protruding spheres 90 Å in diameter. The purified F₁-ATPases have a molecular weight of 320,000 to 400,000 daltons and are composed of five non-identical subunits (, , , and ). The stoichiometry of these subunits in the complex is still unknown but compositions of the type ₃₃ and ₂₂₂₂₂ were found to be consistent with some of the available experimental data. This review discusses the recent data and the experimental approaches utilized for the structural characterization of F₁-ATPases.     

Kinetic studies of ATP synthase: The case for the positional change mechanism

The mitochondrial ATP synthases shares many structural and kinetic properties with bacterial and chloroplast ATP synthases. These enzymes transduce the energy contained in the membrane's electrochemical proton gradients into the energy required for synthesis of high-energy phosphate bonds. The unusual three-fold symmetry of the hydrophilic domain, F₁, of all these synthases is striking. Each F₁ has three identical subunits and three identical subunits as well as three additional subunits present as single copies. The catalytic site for synthesis is undoubtedly contained in the subunit or an , interface, and thus each enzyme appears to contain three identical catalytic sites. This review summarizes recent isotopic and kinetic evidence in favour of the concept, originally proposed by Boyer and coworkers, that energy from the proton gradient is exerted not directly for the reaction at the catalytic site, but rather to release product from a single catalytic site. A modification of this binding change hypotheses is favored by recent data which suggest that the binding change is due to a positional change in all three subunits relative to the remaining subunits of F₁ and F₀ and that the vector of rotation is influenced by energy. The positional change, or rotation, appears to be the slow step in the process of catalysis and it is accelerated in all F₁F₀ ATPases studied by substrate binding and by the proton gradient. However, in the mammalian mitochondrial enzyme, other types of allosteric rate regulation not yet fully elucidated seem important as well.     

Inhibitory Mg-ADP—Fluoroaluminate Complexes Bound to Catalytic Sites of F1-ATPases: Are They Ground-State or Transition-State Analogs?

Schemes are proposed for coupling sequential opening and closing the three catalytic sites of F₁ to rotation of the subunit during ATP synthesis and hydrolysis catalyzed by the F_oF₁-ATP synthase. A prominent feature of the proposed mechanisms is that the transition state during ATP synthesis is formed when a catalytic site is in the process of closing and that the transition state during ATP hydrolysis is formed when a catalytic site is in the process of opening. The unusual kinetics of formation of Mg-ADP—fluoroaluminate complexes in one or two catalytic sites of nucleotide-depleted MF₁ and wild-type and mutant ₃₃ subcomplexes of TF₁ are also reviewed. From these considerations, it is concluded that Mg-ADP—fluoroaluminate complexes formed at catalytic sites of isolated F₁-ATPases or F₁ in membrane-bound F_oF₁ are ground-state analogs.     

The αβ complexes of ATP synthase: the α3β3 oligomer and α1β1 protomer

The basic structures of the catalytic portion (F₁, ₃₃) of ATP synthase are the ₃₃ hexamer (oligomer with cooperativity) and ₁₁ heterodimer (protomer). These were reconstituted from the and subunits of thermophilic F₁ (TF₁), and the ₃₃ hexamer was crystallized. On electrophoresis, both the dimer and hexamer showed bands with ATPase activity. Using the dimer and hexamer, we studied the nucleotide-dependent rapid molecular dynamics. The formation of the hexamer required neither nucleotide nor Mg. The hexamer was dissociated into the dimer in the presence of MgADP, while the dimer was associated into the hexamer in the presence of MgATP. The hexamer, like mitochondrial F₁ and TF₁, showed two kinds of ATPase activity: one was cooperative and was inhibited by only one BzADP per hexamer, and the other was inhibited by three BzADP per hexamer.     

ATP synthases: insights into their motor functions from sequence and structural analyses

ATP synthases are motor complexes comprised of F₀ and F₁ parts that couple the proton gradient across the membrane to the synthesis of ATP by rotary catalysis. Although a great deal of information has been accumulated regarding the structure and function of ATP synthases, their motor functions are not fully understood. For this reason, we performed the alignments and analyses of the protein sequences comprising the core of the ATP synthase motor complex, and examined carefully the locations of the conserved residues in the subunit structures of ATP synthases. A summary of the findings from this bioinformatic study is as follows. First, we found that four conserved regions in the sequence of subunit are clustered into three patches in its structure. The interactions of these conserved patches with the and subunits are likely to be critical for energy coupling and catalytic activity of the ATP synthase. Second, we located a four-residue cluster at the N-terminal domain of mitochondrial OSCP or bacterial (or chloroplast) subunit which may be critical for the binding of these subunits to F₁. Third, from the localizations of conserved residues in the subunits comprising the rotors of ATP synthases, we suggest that the conserved interaction site at the interface of subunit c and (mitochondria) or (bacteria and chloroplasts) may be important for connecting the rotor of F₁ to the rotor of F₀. Finally, we found the sequence of mitochondrial subunit b to be highly conserved, significantly longer than bacterial subunit b, and to contain a shorter dimerization domain than that of the bacterial protein. It is suggested that the different properties of mitochondrial subunit b may be necessary for interaction with other proteins, e.g., the supernumerary subunits.     

Intracellular sites of the synthesis of sweet potato mitochondrial F1ATPase subunits

Summary Five subunits (-, -, -, - and -subunits) of the six -and -subunits) in the F₁ portion (F₁ATPase) of sweet potato (Ipomoea batatas) mitochondrial adenosine triphosphatase were isolated by an electrophoretic method. The - and -subunits were not distinguishable immunologically but showed completely different tryptic peptide maps, indicating that they were different molecular species. In vitro protein synthesis with isolated sweet potato root mitochondria produced only the -subunit when analyzed with anti-sweet potato F₁ATPase antibody reacting with all the subunits except the -subunit. Sweet potato root poly(A)⁺RNA directed the synthesis of six polypeptides which were immunoprecipitated by the antibody: two of them immunologically related to the -subunit and the others to the - and -subunits. We conclude that the -subunit of the F₁ATPase is synthesized only in the mitochondria and the -, - and -subunits are in the cytoplasm.     

ATP Synthases in the Year 2000: Evolving Views about the Structures of These Remarkable Enzyme Complexes

This introductory article briefly summarizes how our views about the structural features ofATP synthases (F₀F₁) have evolved over the past 30 years and also reviews some of our currentviews in the year 2000 about the structures of these remarkably unique enzyme complexes.Suffice it to say that as we approach the end of the first year of this new millinium, we canbe conservatively confident that we have a reasonably good grasp of the overall low-resolutionstructural features of ATP synthases. Electron microscopy techniques, combined with the toolsof biochemistry, molecular biology, and immunology, have played the leading role here byidentifying the headpiece, basepiece, central stalk, side stalk, cap, and in the mitochondrialenzyme, the collar around the central stalk. We can be reasonably confident also that we havea fairly good grasp of much of the high-resolution structural features of both the F₁ moietycomprised of fives subunit types (, , , , and ) and parts of the F₀ moiety comprised ofeither three (E. coli) or at least ten (mitochondria) subunit types. This information acquiredin several different laboratories, either by X-ray crystallography or NMR spectroscopy, includesdetails about the active site and subunit relationships. Moreover, it is consistent with recentlyreported data that the F₁ moiety may be an ATP driven motor, which, during ATP synthesis,is driven in reverse by the electrochemical proton gradient generated by the electron transportchain. The real structural challenges of the future are to acquire at high resolution completeATP synthase complexes representative of different stages of the catalytic cycle during ATPsynthesis and representative also of key regulatory states.     

The oligomycin sensitivity conferring protein (OSCP) of beef heart mitochondria: Studies of its binding to F1 and its function

The binding of oligomycin sensitivity conferring protein (OSCP) to soluble beef-heart mitochondrial ATPase (F₁) has been investigated. OSCP forms a stable complex with F₁, and the F₁ · OSCP complex is capable of restoring oligomycin- and DCCD-sensitive ATPase activity to F₁- and OSCP-depleted submitochondrial particles. The F₁ · OSCP complex retains 50% of its ATPase activity upon cold exposure while free F₁ is inactivated by 90% or more. Both free F₁ and the F₁ · OSCP complex release upon cold exposure a part—probably 1 out of 3—of their subunits; whether subunits are also lost is uncertain. The cold-treated F₁ · OSCP complex is still capable of restoring oligomycin- and DCCD-sensitive ATPase activity to F₁- and OSCP-depleted particles. OSCP also protects F₁ against modification of its subunit by mild trypsin treatment. This finding together with the earlier demonstration that trypsin-modified F₁ cannot bind OSCP indicates that OSCP binds to the subunit of F₁ and that F₁ contains three binding sites for OSCP. The results are discussed in relation to the possible role of OSCP in the interaction of F₁ with the membrane sector of the mitochondrial ATPase system.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - OSCP oligomycin sensitivity conferring protein - SDS sodium dodecylsulfate This paper is dedicated to the memory of David E. Green—scholar, pioneer, visionary.     

The energy transmission in ATP synthase: From the γ-c rotor to the α3β3 oligomer fixed by OSCP-b stator via the βDELSEED sequence

ATP synthase (F₀F₁) is driven by an electrochemical potential of H⁺ (H⁺). F₀F₁ is composed of an ion-conducting portion (F₀) and a catalytic portion (F₁). The subunit composition of F₁ is ₃₃. The active ₃₃ oligomer, characterized by X-ray crystallography, has been obtained only from thermnophilic F₁ (TF₁). We proposed in 1984 that ATP is released from the catalytic site (C site) by a conformational change induced by the DELSEED sequence via -F₀. In fact, cross-linking of DELSEED to stopped the ATP-driven rotation of in the center of ₃₃. The torque of the rotation is estimated to be 420 pN·å from the H⁺ and H⁺-current through F₀F₁. The angular velocity () of is the rate-limiting step, because H⁺ increased theV _max of H⁺ current through F₀, but not theK _m (ATP). The rotational unit of F₀ (=ab₂c₁₀) is /5, while that in ₃₃ is 2/3. This difference is overcome by an analog-digital conversion via elasticity around DELSEED with a threshold to release ATP. The distance at the C site is about 9.6 å (2,8-diN₃-ATP), and tight Mg-ATP binding in ₃₃ was shown by ESR. The rotational relaxation of TF₁ is too rapid (=100 nsec), but the rate of AT(D)P-induced conformational change of ₃₃ measured with a synchrotron is close to . The ATP bound between the P-loop and E188 is released by the shift of DELSEED from RGL. Considering the viscosity resistance and inertia of the free rotor (-c), there may be a stator containing OSCP (= of TF₁) and F₀-d to hold free rotation of ₃₃.     

Identification of subunits required for the catalytic activity of the F1-ATPase

F₁() complexes containing equimolar ratios of the and subunits have been shown to function as active ATPases, whereas individually isolated and subunits show no real ATPase activity. These results indicate that the single-copy subunits are not required for F₁-ATPase activity. The minimal F₁()-core complexes exhibit, however, lower rates and some different properties from those of their parent whole F₁ or ₃₃ complexes. It is therefore concluded that for obtaining a full spectrum of the characteristic functional properties of an F₁-ATPase the presence of the F₁- subunit is also required. The implications of these findings on the subunit location of both catalytic and noncatalytic nucleotide binding sites is discussed.     

Isolation and characterization of the mitochondrial ATP synthase fromChlamydomonas reinhardtii. cDNA sequence and deduced protein sequence of the α subunit

We have isolated the F₀F₁-ATP synthase complex from oligomycin-sensitive mitochondria of the green algaChlamydomonas reinhardtii. A pure and active ATP synthase was obtained by eans of sonication, extraction with dodecyl maltoside and ion exchange and gel permeation chromatography in the presence of glycerol, DTT, ATP and-21. The enzyme consists of 14 subunits as judged by SDS-PAGE. A cDNA clone encoding the ATP synthase subunit has been sequenced. The deduced protein sequence contains a presequence of 45 amino acids which is not present in the mature protein. The mature protein is 58–70% identical to corresponding mitochondrial proteins from other organisms. In contrast to the ATP synthase subunit fromC. reinhardtii (Franzen and Falk, Plant Mol Biol 19 (1992) 771–780), the protein does not have a C-terminal extension. However, the N-terminal domain of the mature protein is 15–18 residues longer than in ATP synthase subunits from other organisms. Southern blot analysis indicates that the protein is encoded by a single-copy gene.Abbreviations DM dodecyl--D-maltoside - OSCP oligomycin sensitivity conferring protein - PMSF phenyl-methylsulfonylfluoride - DTT dithiothreitol - EDTA ethylenediaminotetraacetic disodium salt     

Interconversion of F430 derivatives of methanogenic bacteria

F₄₃₀ is the prosthetic group of the methylcoenzyme M reductase of methanogenic bacteria. The compound isolated from Methanosarcina barkeri appears to be identical to the one obtained from the only distinctly related Methanobacterium thermoautotrophicum. F₄₃₀ is thermolabile and in the presence of acetonitrile or C10 _in4 ^sup- two epimerization products are obtained upon heating; in the absence of these compounds F₄₃₀ is oxidized to 12, 13-didehydro-F₄₃₀. The latter is stereoselectively reduced under H₂ atmosphere to F₄₃₀ by cell-free extracts of M. barkeri or M. thermoautotrophicum. H₂ may be replaced by the reduced methanogenic electron carrier coenzyme F₄₂₀.Abbreviations CH₃S-CoM methylcoenzyme M, 2-methylthioethanesulfonic acid - HS-CoM coenzyme M, 2-mercaptoethanesulfonic acid - F₄₃₀ Ni(II) tetrahydro-(12, 13)-corphin with a uroporphinoid (III) ligand skeleton - 13-epi-F₄₃₀ and 12,13-di-epi-F₄₃₀ the 12, 13- and 12, 13-derivatives of F₄₃₀ - 12, 13-didehydro-F₄₃₀ F₄₃₀ oxidized at C-12 and C-13 - coenzyme F₄₂₀ 7,8-didemethyl-8-hydroxy-5-deazaflavin derivative - coenzyme F₄₂₀H₂ reduced coenzyme F₄₂₀ - MV⁺ methylviologen semiquinone - HPLC high-performance liquid chromatography     

What Is the Role of ε in the Escherichia coli ATP Synthase?

The ATP synthase from Escherichia coli is a prototype of the ATP synthases that are found in many bacteria, in the mitochondria of eukaryotes, and in the chloroplasts of plants. It contains eight different types of subunits that have traditionally been divided into F₁, a water-soluble catalytic sector, and F_o, a membrane-bound ion transporting sector. In the current rotary model for ATP synthesis, the subunits can be divided into rotor and stator subunits. Several lines of evidence indicate that is one of the three rotor subunits, which rotate through 360 degrees. The three-dimensional structure of is known and its interactions with other subunits have been explored by several approaches. In light of recent work by our group and that of others, the role of in the ATP synthase from E. coli is discussed.