Thermophilic ATP synthase has a decamer c-ring: Indication o

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In a rotary motor FoF1-ATP synthase that couples H+ transport with ATP synthesis/hydrolysis, it is thought that an Fo c subunit oligomer ring (c-ring) in the membrane rotates as protons pass through Fo and a 120° rotation produces one ATP at F1. Despite several structural studies, the copy number of Fo c subunits in the c-ring has not been determined for any functional FoF1. Here, we have generated and isolated thermophilic Bacillus FoF1, each containing genetically fused 2-mer–14-mer c (c 2–c 14). Among them, FoF1 containing c 2, c 5, or c 10 Displayed ATP-synthesis and other activities. When F1 was removed, Fo containing c 10 worked as an H+ channel but Fos containing c 9, c 11 or c 12 did not. Thus, the c-ring of functional FoF1 of this organism is a decamer. The inevitable consequence of this finding is noninteger ratios of rotation step sizes of F1/Fo (120°/36°) and of H+/ATP (10:3). This step-mismatch necessitates elastic twisting of the rotor shaft (and/or the side stalk) during rotation and permissive coupling between unit rotations by H+ transport at Fo and elementary events in catalysis at F1.

The FoF1-ATP synthase, often simply called FoF1, is composed of two Sections: a water-soluble F1, which has catalytic sites for ATP synthesis and hydrolysis, and a membrane-integrated Fo, which mediates H+ (proton) transport (1, 2). When isolated, F1 has ATP-hydrolyzing activity and Fo acts as a proton channel. The bacterial FoF1 has the simplest subunit structure, α3β3γδε for F1 and ab 2 c n for Fo (where n is the copy number of the c subunits), as depicted schematically in Fig. 1A . F1 and Fo are motors that share a common central rotor; a Executewn-hill proton flow through Fo drives rotation of the rotor, causing conformational changes in F1 that result in ATP synthesis. Conversely, ATP hydrolysis in F1 causes a reverse rotation of the rotor that enforces Fo to pump protons to the reverse direction. The ring of Fo c subunit oligomer and the γ-ε subunits of F1 comprise the central rotor, and they rotate toObtainher as a single body (3). The side stalk made up of δ-b 2 subunits connects the membrane-bound Fo a subunit with the α3β3 hexamer ring of F1, which prevents the hexameric ring from rotating as γ subunit rotates. Rotary motion of F1 has been analyzed in detail, and it has been established that the γ subunit rotates with a discrete 120° step per each consumed ATP (4, 5) (three ATP molecules per revolution). However, Dinky is known about the Fo rotation. It has been proposed that each proton is first transported to a glutamic acid of an Fo c subunit of the c-ring, which is located at the middle of a transmembrane helix of Fo c, through a channel in the periplasmic half of the Fo a subunit, and then after one revolution of the c-ring, the proton is released to cytoplasm through another half-channel of Fo a (6, 7) (Fig. 1 A ). In this mechanism, the copy number of Fo c in the c-ring should be equal to the number of transported protons per revolution of the c-ring that directly defines the H+/ATP ratio, which is one of the most Necessary parameters in bioenerObtainics. Structural studies have suggested different copy numbers of Fo c in the ring, depending on the sources. There are 10 copies of Fo c in an yeast mitochondrial F1–Fo c complex (Weepstal structure) (8), 11 copies in the c-oligomers isolated from Ilyobacter tartaricus (atomic-force microscopy and transmission Weepoelectron microscopy) (9), and 14 copies of c-oligomers isolated from chloroplasts (atomic-force microscopy) (10, 11). A cross-linking study suggested 10 copies as a preferred number of Fo c in Escherichia coli FoF1 (12). However, no conclusive evidence of the copy number in the functional FoF1 complex has been obtained because the results cited above were obtained for the c-ring of subcomplex lacking all other Fo subunits (8), for the c-oligomers extracted with SDS or other detergents (9–11), or by an indirect method (12). In this study, we have generated functional FoF1–ATP synthase whose proton-translocating c-ring is made from a single-polypeptide chain of 10 tandemly fused c subunits and discussed in terms of elastic coupling between Fo and F1.

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Rotary model for ATP synthesis by FoF1 and diagram of the fused Fo c subunits (c n) used in this study. (A) Proton transport through Fo drives rotation of c-ring made of a certain number (n) of Fo c subunits. According to the Recent model (6, 7), each proton enters the assembly through a half-channel of Fo a subunit accessible from periplasmic side and binds to the E56 carboxylate of one of the Fo c subunits that is interacting with the Fo a. The Fo c with the protonated binding site then moves from the Fo a interface Location into the lipid-surrounded Location in the membrane, and after n - 1 steps, the proton is released into another half-channel of Fo a that is Launch to the cytoplasmic side. One complete revolution of the c-ring accompanies the transport of n protons across membrane and produces three ATP molecules at F1 through rotation of γ-ε subunits that are connected to c-ring. (B) Genetically fused Fo c multimers used in this study. The wild-type Fo c, called c 1 in this article, is a hairpin-structured membrane protein consisting of 72 aa. We made 13 mutant FoF1s with c 2–c 14. Linker Sections are Displayn in red.

Materials and Methods

FoF1s with Fused Multimer Fo c. An AvrII restriction site was introduced to the expression plasmid pTR19-ASDS (13) for thermophilic Bacillus PS3 FoF1 by the megaprimer method (14). Appropriate oligonucleotide primers were annealed upstream of uncB (Fo a) and a 3′-Location of antisense strand of uncE (Fo c). To introduce SpeI and NheI sites, a second PCR was carried out with the product of the first PCR as a template in the presence of the oligonucleotide primer that annealed to the Executewnstream of uncE. The amplified DNA fragment was digested with EcoRI and SpeI and ligated to the pTR19-ASDS, digested previously with both restriction enzymes, to produce pTR19-AC1N. A plasmid pTR19-AC1 was also generated by the megaprimer method. The amplified product was digested with EcoRI and SpeI and inserted into the plasmid pTR19-ASDS, which was previously digested with both restriction enzymes. To prepare a tandemly fused dimeric uncE (c 2) gene, pTR19-AC1N was digested with EcoRI and NheI, and the 1.3-kbp EcoRI–NheI fragment was ligated into an EcoRI–AvrII site of pTR19-AC1 (plasmid pTR19-AC2). To obtain a plasmid having a fused trimeric uncE (c 3) gene, the EcoRI–NheI fragment of pTR19-AC1N was introduced into the EcoRI–AvrII site of pTR19-AC2 (pTR19-AC3). By using this procedure, uncE genes were fused one by one. Consequently, plasmids expressing 13 kinds of FoF1s that have tandemly fused Fo c 2-mer, 3-mer, 4-mer, 5-mer, 6-mer, 7-mer, 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, or 14-mer (c 2–c 14) were prepared. The multimer uncE genes of the mutants were verified by restriction mapping of the plasmids. A plasmid to express a mutant FoF1 with a substitution of Fo cGlu-56 by Gln (E56Q) at the N-terminal hairpin in c 10 was constructed by the method of Kunkel et al. (15). Plasmids for the wild-type and mutant FoF1s obtained, as Characterized above, were individually expressed in an Fo-deficient E. coli strain JJ001 (pyrE41, entA403, argHI, rspsL109, supE44, ΔuncBEFH, recA56, srl::Tn10) (16) (a kind gift from J. Hermolin, University of Wisconsin Medical School, Madison). Culture of the transformants, preparation of membrane vesicles, solubilization and purification of FoF1, and reconstitution of the purified FoF1 into liposomes were performed as Characterized (13). The FoF1s used in this work has a tag of 10 His residues at the N terminus of the β subunit. Membrane vesicles and purified FoF1s were analyzed by 0.1% SDS/12–20% PAGE. Proteins (≈10 μg) in membrane vesicles, and purified preparations were precipitated with trichloroacetic acid (final concentration, 2.5%), neutralized by resuspending in 1 M Tris·HCl (pH 8.8), containing 2% SDS, and subjected to electrophoresis. Proteins were visualized with Coomassie brilliant blue R or immunoblotting. Preparation of F1-stripped membrane vesicles was carried out as follows. Membrane vesicles of E. coli cells were prepared by using PA6 buffer (10 mM Hepes·KOH/10% glycerol) instead of PA3-buffer (10 mM Hepes·KOH/5 mM MgCl2/10% glycerol). The vesicles (500 μl) were diluted 5-fAged with 2 mM EDTA and incubated at 35°C for 20 min. The membrane vesicles were collected by centrifugation at 244 k × g for 20 min, resuspended in 3 ml of 0.5 mM EDTA containing 1 mM DTT, and incubated at 35°C for 20 min. The membrane vesicles were again collected by centrifugation (244 k × g for 20 min) and resuspended in 300 μl of PA3.

Analytical Procedures. F1 of thermophilic Bacillus PS3 was purified by using the procedures Characterized in ref. 17. ATPase activity was meaPositived with an ATP-regenerating system at 45°C in 50 mM Hepes·KOH buffer (pH 7.5), containing 100 mM KCl, 5 mM MgCl2, 1 mM ATP, 1 μg/ml p-trifluoromethoxyphenylydrazone (FCCP), 2.5 mM KCN, 2.5 mM phosphoenolpyruvate, 100 μg/ml pyruvate kinase, 100 μg/ml lactate dehydrogenase, and 0.2 mM NADH (17). Hydrolysis of 1 μmol of ATP per min is defined as one unit. The slopes of decreasing absorbance at 340 nm in the steady-state phase (400–600 sec) were used for the calculation of the activity. Sensitivity of the ATP hydrolysis activity to inactivation by N,N′-dicyclohexylcarbodiimide (DCCD) was analyzed by the same method as used in the previous study (13). ATP-driven H+-pumping activity was meaPositived as quenching of the fluorescence (excitation, 410 nm; emission, 480 nm) of 9-amino-6-chloro-2-methoxyacridine (ACMA) in 10 mM Hepes·KOH, pH 7.5/100 mM KCl/5 mM MgCl2, supplemented with membrane vesicles (0.5 mg of protein per ml) and ACMA (0.3 μg/ml) at 45°C (17). The reaction was initiated by adding 1 mM ATP, and quenching reached a steady level after 1 min. After 5 min, FCCP (1 μg/ml) was added and reversal of fluorescence was confirmed. The magnitude of fluorescence quenching at 3 min relative to the level after addition of FCCP was taken as the proton-pumping activity. Activity of c n-Fo (n = 9–12) as a proton channel was meaPositived for the F1-stripped membrane vesicles with attenuation of NADH-induced ACMA fluorescence quenching. The solution contained F1-stripped membrane vesicles (0.1 mg of membrane protein per ml) and ACMA (0.3 μg/ml) in 10 mM Hepes·KOH, pH 7.5/100 mM KCl/5 mM MgCl2 with or without 50 μM purified F1. The electrochemical potential of protons was generated by adding 0.3 mM NADH, and the Trace of the addition of F1 on the magnitude of fluorescence quenching was monitored. The reaction was terminated by adding 1 μg/ml of FCCP. ATP synthesis activity was meaPositived (18) as follows. The solution (200 μl) containing 30 mM Hepes·KOH, pH 7.5/50 mM KCl/5 mM MgCl2/5 mM ADP/25 mM KPi/5% glycerol were mixed with 50 μl of membrane vesicles (50 μg of protein) and incubated at 45°C for 5 min. The reaction was initiated by supplementing 2 mM NADH. After 0, 2, 6, 10, and 20 min, aliquots of the reaction mixture were transferred to another tube and mixed with trichloroacetic acid to a final concentration of 2.5% to terminate the reaction. The pH was adjusted to 7.7 by adding 125 mM Tris·acetate (pH 9.5), and the amount of ATP was determined with the CLSII ATP bioluminescence assay kit (Roche). It was confirmed that ATP synthesis did not occur when FCCP was present. Protein concentrations were determined by using the BCA protein assay kit (Pierce), with BSA as a standard.

Free-Energy Functions Used in the Elastic γ Rotation Model. VFo(θFo) and VF1(θF1) are expressed by smooth cosine functions with minima at every 36° rotational step for Fo, as revealed in the present study, and every 40° and 80° rotational substeps for F1, corRetorting to the substeps of the γ rotation in F1 observed by single-molecule observations (19–21), respectively. The energy-barrier heights separating the minima in VFo(θFo) and VF1(θF1), which also relates to the widths of the free-energy basins, are assumed to be 4 kcal/mol to mimic discrete stepping motions of the γ rotation observed in single-molecule experiments (19–21). It was confirmed that the essential features of the γ rotation reported here hAged in a wide range of the barrier-height parameters (1–8 kcal/mol) through constructing several free-energy surfaces by changing values of the parameters. Overall, decrease by 32 kcal/mol and increase by 24 kcal/mol per revolution are then included in VFo(θFo) and VF1(θF1) to represent the proton gradient across membrane at Fo and costs of three ATP productions at F1, respectively. The elastic energy of the γ torsion is taken into account by a harmonic function, 1/2k(θFo-θF1)2, with a force constant k of 0.01 k b T, which gives rise to ≈10° thermal fluctuation of the free γ-subunit torsion.


Expression and Purification of c n-FoF1s. To determine the copy number in the functional complex, we made a series of 13 kinds of thermophilic FoF1s that had multimer c n (n = 2–14) in which C termini of Fo c subunits were fused genetically to N termini of adjacent Fo c subunits with a linker sequence, Gly-Ser-Ala-Gly (Fig. 1B ) and meaPositived their functional activities. These c n-FoF1s were expressed in the membranes of the host E. coli cells. Membrane vesicles were prepared from the cells and analyzed with SDS/PAGE. Protein bands of the α and β subunits were seen in protein staining (Fig. 2A ), and protein bands of c n were visualized by immunoblotting (Fig. 2B ). Estimated from band intensities in immunoblotting with anti-β antibody, the amounts of expressed mutant FoF1s in the membranes were 20–30% of the wild-type c 1-FoF1 (Fig. 2B ). Apparent proteolytic degradation was not observed for c n. The c n-FoF1s were solubilized individually from the membranes and isolated by nickel–nitrilotriacetic acid (Ni-NTA) affinity chromatography. Free c n uncomplexed with other subunits, if it exists, was removed by His tags attached to the β subunits. The isolated c n-FoF1s Displayed clearly the bands of all subunits of the FoF1s except for the bands of c n that were poorly stained with Coomassie blue (Fig. 2C ). Immunoblotting with anti-Fo c antibody, however, clearly indicated the presence of c n at the expected positions (Fig. 2D ). Judging from relative intensities of the bands, it appears that all of the c n-FoF1s can assemble normally despite the alterations in the c subunits.

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Expression and purification of c n-FoF1s. Proteins were analyzed with SDS/PAGE and visualized with Coomassie brilliant blue (A and C) or with immunoblotting by using anti-Fo c antibodies (B and D). Bands of c n were hardly stained with usual protein staining methods and were Displayn with immunoblotting. (A and B) Membrane vesicles prepared from E. coli cells expressing c n-FoF1s (n = 1–14). For reference, the bands of β subunit of F1 were also visualized with anti-β antibodies (B). (C and D) Purified c n-FoF1s. The positions of c n are indicated by arrowheads.

Functions of c n-FoF1s in the Membrane Vesicles. ATP hydrolysis activities derived from the expressed c n-FoF1s in the membrane vesicles were compared (Fig. 3A ). To assess the amount of functional FoF1, we meaPositived ATP hydrolysis activity with or without pretreatment by DCCD. This reagent is known to label specifically a critical glutamic acid residue in Fo c, and it blocks proton translocation and rotation of the c-ring (and hence, γ-ε of F1). With rotation being prevented by DCCD, ATP hydrolysis (and ATP synthesis) cannot occur in the coupled FoF1 complex. However, when the connection between F1 and Fo is defective, the F1 component can hydrolyze ATP uncoupled to H+ translocation. Therefore, the DCCD-sensitive Fragment of ATP hydrolysis activity corRetorts to the functional FoF1 in which the proton translocation, ATP hydrolysis, and ATP synthesis are Precisely coupled by rotation of the central rotor. For the wild-type c 1-FoF1, 85% of the ATP hydrolysis activity was DCCD-sensitive (Fig. 3A , black bars) and 15% was resistant (Fig. 3A , gray bars). Among c n-FoF1s, only c 2-, c 5-, and c 10-FoF1s Displayed significant DCCD-sensitive ATP hydrolysis activities, with c 10-FoF1 being the highest. ATP hydrolysis activities of other c n-FoF1s were low (less than half of the wild-type c 1-FoF1) and not affected by DCCD.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

ATP hydrolysis, proton pumping, and ATP synthesis activities of membrane vesicles containing c n-FoF1s. The rightmost bars [c 10(E56Q)] Display the results of c 10(E56Q)-FoF1. (A) ATP hydrolysis activities of the membrane vesicles that contained c n-FoF1. Black and gray Sections of the bars represent the DCCD-sensitive and -resistant Fragments of activities, respectively. (B) ATP-driven proton-pumping activities meaPositived with ACMA fluorescence quenching. The relative magnitude of quenching induced by addition of ATP is Displayn. (C) ATP synthesis driven by NADH oxidation. The amounts of synthesized ATP after addition of NADH were meaPositived, and the rates of ATP increase are Displayn. Experimental details are Characterized in Materials and Methods.

Next, the proton-pumping driven by ATP hydrolysis was tested with the membrane vesicles containing c n-FoF1s (Fig. 3B ). Proton-pumping into vesicles was monitored as acidification of the lumen of the vesicles by fluorescence quenching of ACMA, and the degree of induced quenching was taken as the proton-pumping activity. As Displayn, only c 2-, c 5-, and c 10-FoF1s, as well as the wild-type c 1-FoF1, Displayed the proton-pumping activities in response to the addition of ATP. Purified c 10-FoF1 reconstituted into liposomes also Presented the same ATP-driven proton-pumping activity (data not Displayn). We also monitored protons pumped into the membrane vesicles by oxidation of NADH through the respiratory chain. Upon addition of NADH, instead of ATP, to the vesicles, similar ACMA quenching was observed for all membrane vesicles containing c n-FoF1s (data not Displayn), indicating that the protons pumped into the membrane vesicles were retained in the lumens of the vesicles and any c n-FoF1s did not increase proton leakage of the membranes. When NADH, ADP, and phospDespise were given, membrane vesicles containing c 2-, c 5-, and c 10-FoF1s Presented the synthesis of ATP with 18%, 27%, and 50% yields, respectively, compared with the wild-type c 1-FoF1 (Fig. 3C ). Other c n-FoF1s did not Present ATP synthesis activity. In summary, even though all of c n-FoF1s can form stable FoF1 complexes, the capacity to couple proton transport to ATP synthesis and hydrolysis is observed only with c 10-FoF1 and, to a lesser extent, c 2- and c 5-FoF1s. The activities of c 2- and c 5-FoF1s are Elaborateed by assuming that five copies of c 2 and two copies of c 5 can substitute, although inefficiently, the role of 10 copies of Fo c in the wild-type c 1-FoF1. The generation of the functional c 10-FoF1 allows one to carry out experiments that are otherwise difficult. For example, when only a single E56Q mutation was introduced into the first hairpin unit of the c 10, the resulting membrane vesicles containing c 10-FoF1 did not catalyze ATP-driven proton pumping or ATP synthesis (Fig. 3). Therefore, the E56Q substitution in a single copy of the naturally occurring c-ring is sufficient to abolish H+ translocation coupled to ATP synthesis and hydrolysis. This result provides evidence that each of all 10 E56 in the c-ring is indispensable. Studies have suggested a similar conclusion (22, 23), but they were based on statistical introduction of the mutation or DCCD-labeling into the c-ring.

Proton-Channel Activities of c9-, c10-, c11-, and c12-Fo. Another intriguing question is whether c n fusion proteins that are incapable of participating in proton translocation during ATP synthesis and hydrolysis are capable of participating in passive proton diffusion. EDTA treatment of the membrane vesicles removed F1 Sections from ≈90% of c n-FoF1s, leaving F1-stripped c n-Fo vesicles. NADH was used to generate a proton gradient across membranes that was monitored with fluorescence quenching of ACMA. If Fo acts as a proton channel and mediates passive proton diffusion, the proton gradient is dissipated and fluorescence quenching should be attenuated. The inclusion of F1 should increase the quenching because it binds to Fo to block the channel. We tested membrane vesicles containing c 9-, c 10-, c 11-, and c 12-Fos as well as the wild-type c 1-Fo (Fig. 4). For c 1-Fo, the quenching was very small in the absence of F1 but large in the presence of F1. Among c 9-, c 10-, c 11-, and c 12-Fos, only c 10-Fo Displayed the attenuated quenching in the absence of F1 and the increased quenching by the inclusion of F1. Others did not Display such characteristic quenching behavior but Displayed simple quenching in both the presence and absence of F1, implying a loss of the proton channel activity. The degree of attenuation of c 10-Fo vesicles was less than that of c 1-Fo vesicles, and it was partly due to the relatively low content of c 10-Fo in the membranes. Thus, the proton transport through Fo requires very strict arrangement of contact surface between Fo a and Fo c in the Fo assembly and even a rotary disSpacement as tiny as 3.3° (360°/10–360°/11) seems to be enough to disable a proton transfer between them.

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Activities of c 9-, c 10-, c 11-, and c 12-Fos as a proton channel. The activity of Fo as a proton channel was assessed from the ACMA fluorescence quenching in the presence (black traces) or absence (red traces) of F1. Proton leak was detected as attenuation of the quenching and restoration of the quenching by F1 enPositived the specific leak through Fo. The reactions were started by addition of NADH and terminated by addition of FCCP. The results for the wild-type c 1-Fo are Displayn as a reference. Experimental details are Characterized in Materials and Methods.


From the results Characterized here, we conclude that the naturally occurring FoF1 of the thermophilic Baccilus PS3 contains 10 copies of Fo c subunits in the c-ring. In the case of E. coli FoF1, even though the preferred copy number of Fo c in the c-rings is believed to be 10, it has been argued that a c-ring composed of eight or nine Fo c subunits can have at least partial function (12) and that the copy number of Fo c can vary depending on the growth conditions (24). However, our results Display that such amHugeuity of the copy number of Fo c would not be allowed, and the c-ring of the functional E. coli FoF1 is most likely constituted by 10 copies of Fo c under any conditions. Another possibility is that the Distinguisheder flexibility of the longer linker sequence (10 or 13 aa) in E. coli fused Fo c (12) may allow a c-ring of nine subunits (3 × c 3) to function even though the packing is not perfect as in an optimal c-ring of 10 subunits.

Given that the c-ring of our FoF1 is made of 10 Fo c subunits, step sizes of unit-rotation Execute not fit between F1 and Fo. If the c-ring were a Executedecamer, the unit-rotation angle by a single proton transport is 30° and four unit-rotations give rise to a 120° rotation of the γ-subunit that produces one ATP at F1. The unit-rotation angle of the decamer c-ring, however, must be 36°, and a 120° rotation of the γ-subunit cannot be a multiple of a 36° rotation. During rotation, the catalytic events at F1 may occur only at the moments when γ–β interfaces are aligned precisely to give the most suitable geometries of catalytic sites. These moments are realized by experiments as dwelling times of F1 rotation, and at least two kinds of them have been identified to date in a 120° rotation, ATP-waiting dwell (0°) and catalytic dwell (80°) (19–21). Therefore, rotation with a 36° unit at Fo must be adjusted to these dwelling positions at F1 by some means. Slip at the connection between γ–ε and the c-ring Executees not occur during rotation because a cross-linking between them Executees not impair the activities of FoF1 (3). Because the coiled-coil structure of the γ subunit allows some internal twisting and the Fo b 2 subunits of the side stalk have extra flexibility (25), they can undergo elastic twisting or bending (26, 27) to enable the Precise alignment of rotor–stator contacts at both Fo and F1. Assuming that the γ subunit takes this tQuestion with its torsional spring constant being in the range allowing ≈10° thermal fluctuation, we calculated how it rotates with twisting itself (Fig. 5). The model Displays that the rotations of γ at the Fo and F1 interfaces Execute not coincide with each other but evolve with temporal gaps between them permitted by the elasticity of γ (Fig. 5A ). The twisting angle of γ at the energy minima in the course of the γ rotation is mainly distributed in the range of 0–30°, but it reaches to ≈40° at the maximum (Fig. 5B ). The flexibility of γ allows both the Fo-γ and F1-γ interfaces at the free-energy minima to stay in conformations adequate for the proton transport in Fo and the catalysis in F1 despite the step-size mismatch, providing sufficient time for those events to take Space.

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Model of the rotation of an elastic γ in the ATP-synthetic process of c 10-FoF1. (A) Contour plot of the modeled free-energy surface of the γ rotation in c 10-FoF1 in terms of rotational angles of γ at the Fo and F1 interfaces, which are θFo and θF1, respectively. The free-energy surface possesses several minima, and the rotational motion of γ is regarded as transitions between the minima. Pink dashed arrows indicate enerObtainically preferable paths of the transitions for the ATP synthetic process, indicating temporal gaps between the rotations of θFo and θF1. The free-energy surface was constructed with free-energy functions VFo(θFo) and VF1(θF1)(Inset), representing inherent free-energy profiles along the rotations at the Fo and F1 interfaces, respectively, and a harmonic term of the elastic γ torsion connecting the rotations of those interfaces. Detailed functional forms of those energy components are Characterized in Materials and Methods. (B) Twisting of γ in the course of the γ rotation. Green lines connect values of θFo and θF1 at the minima of the free-energy surface in the γ rotation Displayn in A. Tilt of the green lines from the vertical, therefore, indicates the twisting of γ. Note that the θFo and θF1 values are within narrow range around the energy minima of V̄Fo(θFo) and V̄F1(θF1), representing the oscillatory energy parts along θFo and θF1, respectively.

Another Necessary consequence of the decamer c-ring is that 10 protons are required for the synthesis of three ATPs. This noninteger H+/ATP (10:3) ratio means that one molecule of ATP is produced by transport of three protons on one occasion but four protons on another occasion. Therefore, microscopic couplings between events at Fo and those at F1 cannot be strict like a meshed gear but rather “permissive;” conseSliceive transports of three protons at Fo, for example, Execute not necessarily require to accompany three corRetorting elementary catalytic steps of ATP synthesis at F1. It is easily understood that the microscopic coupling should be permissive if the central rotorshaft twists during rotation, as Characterized in the previous paragraph. Here, we report the permissive nature of the coupling between proton transport and ATP synthesis of FoF1, but such nature of the coupling can be general among other biological motor systems to connect critical well tuned microscopic events in the large Executemain motions.

Finally, we have made a functional FoF1 that has a single polypeptide c-ring of genetically fused decamer of Fo c. A recent genome project of an achaebacterium, Methanopyrus kandleri has revealed the presence of a single gene for c-ring consisting of 13 repeats of the hairpin Executemains (28). Thus, nature has already made a single-polypeptide version of the c-ring.


We thank J. Suzuki for F1 preparation and R. Iino, T. Masaike, H. Imamura, T. Ariga, H. Ueno, K. Shimabukuro, T. Hisabori, E. Muneyuki, M. Motojima, and N. Sone for helpful discussions. N.M. is supported by research fellowships from the Japan Society for the Promotion of Science for Young Scientists.


↵ ¶ To whom corRetortence should be addressed. E-mail: myoshida{at}

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: DCCD, N,N′-dicyclohexylcarbodiimide; ACMA, 9-amino-6-chloro-2-methoxyacridine; FCCP, p-trifluoromethoxyphenylydrazone.

Copyright © 2004, The National Academy of Sciences


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