Half channels mediating H transport and the mechanism of gating in the Fo sector of Escherichia coli F1Fo ATP synthase

H⁺-transporting F₁F_o ATP synthase catalyzes the synthesis of ATP via coupled rotary motors within F_o and F₁. H⁺ transport at the subunit a–c interface in trans-membranous F_o drives rotation of the c-ring within the membrane, with subunit c being bound in a complex with the γ and ε subunits extending from the membrane. Finally, the rotation of subunit γ within the α₃β₃ sector of F₁ mechanically drives ATP synthesis within the catalytic sites. In this review, we propose and provide evidence supporting the route of proton transfer via half channels from one side of the membrane to the other, and the mechanism of gating H⁺ binding to and release from Asp61 of subunit c, via conformational movements of Arg210 in subunit a. We propose that protons are gated from the inside of a four-helix bundle at the periplasmic side of subunit a to drive protonation of cAsp61, and that this gating movement is facilitated by the swiveling of trans-membrane helices (TMHs) 4 and 5 at the site of interaction with cAsp61 on the periphery of the c-ring. Proton release to the cytoplasmic half channel is facilitated by the movement of aArg210 as a consequence of this proposed helical swiveling. Finally, release from the cytoplasmic half channel is mediated by residues in a complex of interacting extra-membraneous loops formed between TMHs of both subunits a and c. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Active-Site Structure of the Thermophilic Foc-Subunit Ring in Membranes Elucidated by Solid-State NMR

F_oF₁-ATP synthase uses the electrochemical potential across membranes or ATP hydrolysis to rotate the F_oc-subunit ring. To elucidate the underlying mechanism, we carried out a structural analysis focused on the active site of the thermophilic c-subunit (TF_oc) ring in membranes with a solid-state NMR method developed for this purpose. We used stereo-array isotope labeling (SAIL) with a cell-free system to highlight the target. TF_oc oligomers were purified using a virtual ring His tag. The membrane-reconstituted TF_oc oligomer was confirmed to be a ring indistinguishable from that expressed in E. coli on the basis of the H⁺-translocation activity and high-speed atomic force microscopic images. For the analysis of the active site, 2D ¹³C-¹³C correlation spectra of TF_oc rings labeled with SAIL-Glu and -Asn were recorded. Complete signal assignment could be performed with the aid of the C^α_i+1-C^α_i correlation spectrum of specifically ¹³C,¹⁵N-labeled TF_oc rings. The C^δ chemical shift of Glu-56, which is essential for H⁺ translocation, and related crosspeaks revealed that its carboxyl group is protonated in the membrane, forming the H⁺-locked conformation with Asn-23. The chemical shift of Asp-61 C^γ of the E. coli c ring indicated an involvement of a water molecule in the H⁺ locking, in contrast to the involvement of Asn-23 in the TF_oc ring, suggesting two different means of proton storage in the c rings.     

The c13 Ring from a Thermoalkaliphilic ATP Synthase Reveals an Extended Diameter Due to a Special Structural Region

We have structurally characterized the c-ring from the thermoalkaliphilic Bacillus sp. strain TA2.A1 F₁F_o-ATP synthase. Atomic force microscopy imaging and cryo-electron microscopy analyses confirm previous mass spectrometric data indicating that this c-ring contains 13 c-subunits. The cryo-electron microscopy map obtained from two-dimensional crystals shows less closely packed helices in the inner ring compared to those of Na⁺-binding c₁₁ rings. The inner ring of α-helices in c₁₁ rings harbors a conserved GxGxGxGxG motif, with glycines located at the interface between c-subunits, which is responsible for the close packing of these helices. This glycine motif is altered in the c₁₃ ring of Bacillus sp. strain TA2.A1 to AxGxSxGxS, leading to a change in c-c subunit contacts and thereby enlarging the c-ring diameter to host a greater number of c-subunits. An altered glycine motif is a typical feature of c-subunit sequences in alkaliphilic Bacillus species. We propose that enlarged c-rings in proton-dependent F-ATP synthases may represent an adaptation to facilitate ATP synthesis at low overall proton-motive force, as occurs in bacteria that grow at alkaline pH.     

Crystallographic structure of the turbine C-ring from spinach chloroplast F-ATP synthase

In eukaryotic and prokaryotic cells, F-ATP synthases provide energy through the synthesis of ATP. The chloroplast F-ATP synthase (CF₁F_O-ATP synthase) of plants is integrated into the thylakoid membrane via its F_O-domain subunits a, b, b’ and c. Subunit c with a stoichiometry of 14 and subunit a form the gate for H⁺-pumping, enabling the coupling of electrochemical energy with ATP synthesis in the F₁ sector.Here we report the crystallization and structure determination of the c14-ring of subunit c of the CF₁F_O-ATP synthase from spinach chloroplasts. The crystals belonged to space group C2, with unit-cell parameters a=144.420, b=99.295, c=123.51 Å, and β=104.34° and diffracted to 4.5 Å resolution. Each c-ring contains 14 monomers in the asymmetric unit. The length of the c-ring is 60.32 Å, with an outer ring diameter 52.30 Å and an inner ring width of 40 Å.     

Active-Site Structure of the Thermophilic Foc-Subunit Ring in Membranes Elucidated by Solid-State NMR

F_oF₁-ATP synthase uses the electrochemical potential across membranes or ATP hydrolysis to rotate the F_oc-subunit ring. To elucidate the underlying mechanism, we carried out a structural analysis focused on the active site of the thermophilic c-subunit (TF_oc) ring in membranes with a solid-state NMR method developed for this purpose. We used stereo-array isotope labeling (SAIL) with a cell-free system to highlight the target. TF_oc oligomers were purified using a virtual ring His tag. The membrane-reconstituted TF_oc oligomer was confirmed to be a ring indistinguishable from that expressed in E. coli on the basis of the H⁺-translocation activity and high-speed atomic force microscopic images. For the analysis of the active site, 2D ¹³C-¹³C correlation spectra of TF_oc rings labeled with SAIL-Glu and -Asn were recorded. Complete signal assignment could be performed with the aid of the C^α_i+1-C^α_i correlation spectrum of specifically ¹³C,¹⁵N-labeled TF_oc rings. The C^δ chemical shift of Glu-56, which is essential for H⁺ translocation, and related crosspeaks revealed that its carboxyl group is protonated in the membrane, forming the H⁺-locked conformation with Asn-23. The chemical shift of Asp-61 C^γ of the E. coli c ring indicated an involvement of a water molecule in the H⁺ locking, in contrast to the involvement of Asn-23 in the TF_oc ring, suggesting two different means of proton storage in the c rings.     

Mutational analysis of a conserved positive charge in the c-ring of E. coli ATP synthase

F₁F_o ATP synthase is a ubiquitous molecular motor that utilizes a rotary mechanism to synthesize adenosine triphosphate (ATP), the fundamental energy currency of life. The membrane-embedded F_o motor converts the electrochemical gradient of protons into rotation, which is then used to drive the conformational changes in the soluble F₁ motor that catalyze ATP synthesis. In E. coli, the F_o motor is composed of a c₁₀ ring (rotor) alongside subunit a (stator), which together provide two aqueous half channels that facilitate proton translocation. Previous work has suggested that Arg50 and Thr51 on the cytoplasmic side of each subunit c are involved in the proton translocation process, and positive charge is conserved in this region of subunit c. To further investigate the role of these residues and the chemical requirements for activity at these positions, we generated 13 substitution mutants and assayed their in vitro ATP synthesis, H⁺ pumping, and passive H⁺ permeability activities, as well as the ability of mutants to carry out oxidative phosphorylation in vivo. While polar and hydrophobic mutations were generally tolerated in either position, introduction of negative charge or removal of polarity caused a substantial defect. We discuss the possible effects of altered electrostatics on the interaction between the rotor and stator, water structure in the aqueous channel, and interaction of the rotor with cardiolipin.     

A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif

The anaerobic bacterium Fusobacterium nucleatum uses glutamate decarboxylation to generate a transmembrane gradient of Na⁺. Here, we demonstrate that this ion-motive force is directly coupled to ATP synthesis, via an F₁F_o-ATP synthase with a novel Na⁺ recognition motif, shared by other human pathogens. Molecular modeling and free-energy simulations of the rotary element of the enzyme, the c-ring, indicate Na⁺ specificity in physiological settings. Consistently, activity measurements showed Na⁺ stimulation of the enzyme, either membrane-embedded or isolated, and ATP synthesis was sensitive to the Na⁺ ionophore monensin. Furthermore, Na⁺ has a protective effect against inhibitors targeting the ion-binding sites, both in the complete ATP synthase and the isolated c-ring. Definitive evidence of Na⁺ coupling is provided by two identical crystal structures of the c₁₁ ring, solved by X-ray crystallography at 2.2 and 2.6 Å resolution, at pH 5.3 and 8.7, respectively. Na⁺ ions occupy all binding sites, each coordinated by four amino acids and a water molecule. Intriguingly, two carboxylates instead of one mediate ion binding. Simulations and experiments demonstrate that this motif implies that a proton is concurrently bound to all sites, although Na⁺ alone drives the rotary mechanism. The structure thus reveals a new mode of ion coupling in ATP synthases and provides a basis for drug-design efforts against this opportunistic pathogen.     

Intersubunit bridging by Na+ ions as a rationale for the unusual stability of the c-rings of Na+-translocating F1F0 ATP synthases

The oligomeric c-rings of Na⁺-translocating F₁F₀ ATP synthases exhibit unusual stability, resisting even boiling in SDS. Here, we show that the molecular basis for this remarkable property is intersubunit crossbridging by Na⁺ or Li⁺ ions. The heat stability of c₁₁ was dependent on the presence of Na⁺ or Li⁺ ions. For equal stability, 10 times higher Li⁺ than Na⁺ concentrations were required, reflecting the 10 times lower binding affinity for Li⁺ than for Na⁺. In a recent structural model of c₁₁, the Na⁺ or Li⁺ binding ligands are located on neighboring c-subunits, which thus become crossbridged by the binding of either alkali ion with a concomitant increase in the stability of the ring. Site-directed mutagenesis strengthens the essential role of glutamate 65 in the crossbridging of the subunits and also corroborates the proposed stabilizing effect of an ion bridge including aspartate 2.     

Functional production of the Na+ F1FO ATP synthase from Acetobacterium woodii in Escherichia coli requires the native AtpI

The Na⁺ F₁F_O ATP synthase of the anaerobic, acetogenic bacterium Acetobacterium woodii has a unique F_OV_O hybrid rotor that contains nine copies of a F_O-like c subunit and one copy of a V_O-like c ₁ subunit with one ion binding site in four transmembrane helices whose cellular function is obscure. Since a genetic system to address the role of different c subunits is not available for this bacterium, we aimed at a heterologous expression system. Therefore, we cloned and expressed its Na⁺ F₁F_O ATP synthase operon in Escherichia coli. A Δatp mutant of E. coli produced a functional, membrane-bound Na⁺ F₁F_O ATP synthase that was purified in a single step after inserting a His₆-tag to its β subunit. The purified enzyme was competent in Na⁺ transport and contained the F_OV_O hybrid rotor in the same stoichiometry as in A. woodii. Deletion of the atpI gene from the A. woodii operon resulted in a loss of the c ring and a mis-assembled Na⁺ F₁F_O ATP synthase. AtpI from E. coli could not substitute AtpI from A. woodii. These data demonstrate for the first time a functional production of a F_OV_O hybrid rotor in E. coli and revealed that the native AtpI is required for assembly of the hybrid rotor.     

Structural and energetic basis for H+ versus Na+ binding selectivity in ATP synthase Fo rotors

The functional mechanism of the F₁F_o ATP synthase, like many membrane transporters and pumps, entails a conformational cycle that is coupled to the movement of H⁺ or Na⁺ ions across its transmembrane domain, down an electrochemical gradient. This coupling is an efficient means of energy transduction and regulation, provided that ion binding to the membrane domain, known as F_o, is appropriately selective. In this study we set out to establish the structural and energetic basis for the ion-binding selectivity of the membrane-embedded F_o rotors of two representative ATP synthases. First, we use a biochemical approach to demonstrate the inherent binding selectivity of these rotors, that is, independently from the rest of the enzyme. We then use atomically detailed computer simulations of wild-type and mutagenized rotors to calculate and rationalize their selectivity, on the basis of the structure, dynamics and coordination chemistry of the binding sites. We conclude that H⁺ selectivity is most likely a robust property of all F_o rotors, arising from the prominent presence of a conserved carboxylic acid and its intrinsic chemical propensity for protonation, as well as from the structural plasticity of the binding sites. In H⁺-coupled rotors, the incorporation of hydrophobic side chains to the binding sites enhances this inherent H⁺ selectivity. Size restriction may also favor H⁺ over Na⁺, but increasing size alone does not confer Na⁺ selectivity. Rather, the degree to which F_o rotors may exhibit Na⁺ coupling relies on the presence of a sufficient number of suitable coordinating side chains and/or structural water molecules. These ligands accomplish a shift in the relative binding energetics, which under some physiological conditions may be sufficient to provide Na⁺ dependence.     

Complementation of the Fo c Subunit of Escherichia coli with That of Streptococcus mutans and Properties of the Hybrid FoF1 ATP Synthase

The c subunit of Streptococcus mutans ATP synthase (F_oF₁) is functionally exchangeable with that of Escherichia coli, since E. coli with a hybrid F_oF₁ is able to grow on minimum succinate medium through oxidative phosphorylation. E. coli F₁ bound to the hybrid F_o with the S. mutans c subunit showed N,N′-dicyclohexylcarbodiimide-sensitive ATPase activity similar to that of E. coli F_oF₁. Thus, the S. mutans c subunit assembled into a functional F_o together with the E. coli a and b subunits, forming a normal F₁ binding site. Although the H⁺ pathway should be functional, as was suggested by the growth on minimum succinate medium, ATP-driven H⁺ transport could not be detected with inverted membrane vesicles in vitro. This observation is partly explained by the presence of an acidic residue (Glu-20) in the first transmembrane helix of the S. mutans c subunit, since the site-directed mutant carrying Gln-20 partly recovered the ATP-driven H⁺ transport. Since S. mutans is recognized to be a primary etiological agent of human dental caries and is one cause of bacterial endocarditis, our system that expresses hybrid F_o with the S. mutans c subunit would be helpful to find antibiotics and chemicals specifically directed to S. mutans.     

The phototroph-specific β-hairpin structure of the γ subunit of FoF1-ATP synthase is important for efficient ATP synthesis of cyanobacteria

The F_oF₁ synthase produces ATP from ADP and inorganic phosphate. The γ subunit of F_oF₁ ATP synthase in photosynthetic organisms, which is the rotor subunit of this enzyme, contains a characteristic β-hairpin structure. This structure is formed from an insertion sequence that has been conserved only in phototrophs. Using recombinant subcomplexes, we previously demonstrated that this region plays an essential role in the regulation of ATP hydrolysis activity, thereby functioning in controlling intracellular ATP levels in response to changes in the light environment. However, the role of this region in ATP synthesis has long remained an open question because its analysis requires the preparation of the whole F_oF₁ complex and a transmembrane proton-motive force. In this study, we successfully prepared proteoliposomes containing the entire F_oF₁ ATP synthase from a cyanobacterium, Synechocystis sp. PCC 6803, and measured ATP synthesis/hydrolysis and proton-translocating activities. The relatively simple genetic manipulation of Synechocystis enabled the biochemical investigation of the role of the β-hairpin structure of F_oF₁ ATP synthase and its activities. We further performed physiological analyses of Synechocystis mutant strains lacking the β-hairpin structure, which provided novel insights into the regulatory mechanisms of F_oF₁ ATP synthase in cyanobacteria via the phototroph-specific region of the γ subunit. Our results indicated that this structure critically contributes to ATP synthesis and suppresses ATP hydrolysis.     

Obstruction of Transmembrane Helical Movements in Subunit a Blocks Proton Pumping by F1Fo ATP Synthase

Subunit a plays a key role in promoting H⁺ transport-coupled rotary motion of the subunit c ring in F₁F_o ATP synthase. H⁺ binding and release occur at Asp-61 in the middle of the second transmembrane helix (TMH) of F_o subunit c. H⁺ are thought to reach cAsp61 via aqueous half-channels formed by TMHs 2–5 of subunit a. Movements of TMH4 and TMH5 have been proposed to facilitate protonation of cAsp61 from a half channel centered in a four helix bundle at the periplasmic side of subunit a. The possible necessity of these proposed TMH movements was investigated by assaying ATP driven H⁺ pumping function before and after cross-linking paired Cys substitutions at the center of TMHs within subunit a. The cross-linking of the Cys pairs aG218C/I248C in TMH4 and TMH5, and aL120C/H245C in TMH2 and TMH5, inhibited H⁺ pumping by 85–90%. H⁺ pumping function was largely unaffected by modification of the same Cys residues in the absence of cross-link formation. The inhibition is consistent with the proposed requirement for TMH movements during the gating of periplasmic H⁺ access to cAsp61. The cytoplasmic loops of subunit a have been implicated in gating H⁺ release to the cytoplasm, and previous cross-linking experiments suggest that the chemically reactive regions of the loops may pack as a single domain. Here we show that Cys substitutions in these domains can be cross-linked with retention of function and conclude that these domains need not undergo large conformational changes during enzyme function.     

Structural model of the transmembrane Fo rotary sector of H-transporting ATP synthase derived by solution NMR and intersubunit cross-linking in situ

H⁺-transporting, F₁F_o-type ATP synthases utilize a transmembrane H⁺ potential to drive ATP formation by a rotary catalytic mechanism. ATP is formed in alternating β subunits of the extramembranous F₁ sector of the enzyme, synthesis being driven by rotation of the γ subunit in the center of the F₁ molecule between the alternating catalytic sites . The H⁺ electrochemical potential is thought to drive γ subunit rotation by first coupling H⁺ transport to rotation of an oligomeric rotor of c subunits within the transmembrane F_o sector. The γ subunit is forced to turn with the c-oligomeric rotor due to connections between subunit c and the γ and ε subunits of F₁. In this essay we will review recent studies on the Escherichia coli F_o sector. The monomeric structure of subunit c, determined by NMR, shows that subunit c folds in a helical hairpin with the proton carrying Asp⁶¹ centered in the second transmembrane helix (TMH). A model for the structural organization of the c₁₀ oligomer in F_o was deduced from extensive cross-linking studies and by molecular modeling. The model indicates that the H⁺-carrying carboxyl of subunit c is occluded between neighboring subunits of the c₁₀ oligomer and that two c subunits pack in a “front-to-back” manner to form the H⁺ (cation) binding site. In order for protons to gain access to Asp⁶¹ during the protonation/deprotonation cycle, we propose that the outer, Asp⁶¹-bearing TMH-2s of the c-ring and TMHs from subunits composing the inlet and outlet channels must turn relative to each other, and that the swiveling motion associated with Asp⁶¹ protonation/deprotonation drives the rotation of the c-ring. The NMR structures of wild-type subunit c differs according to the protonation state of Asp⁶¹. The idea that the conformational state of subunit c changes during the catalytic cycle is supported by the cross-linking evidence in situ, and two recent NMR structures of functional mutant proteins in which critical residues have been switched between TMH-1 and TMH-2. The structural information is considered in the context of the possible mechanism of rotary movement of the c₁₀ oligomer during coupled synthesis of ATP.     

Microsecond motions probed by near-rotary-resonance <Emphasis Type="Italic">R</Emphasis><Subscript>1ρ</Subscript><Superscript>15</Superscript>N MAS NMR experiments: the model case of protein overall-rocking in crystals

F_oF₁-ATP synthase catalyzes ATP hydrolysis/synthesis coupled with a transmembrane H⁺ translocation in membranes. The F_o c-subunit ring plays a major role in this reaction. We have developed an assignment strategy for solid-state ¹³C NMR (ssNMR) signals of the F_o c-subunit ring of thermophilic Bacillus PS3 (TF_o c-ring, 72 residues), carrying one of the basic folds of membrane proteins. In a ssNMR spectrum of uniformly ¹³C-labeled sample, the signal overlap has been a major bottleneck because most amino acid residues are hydrophobic. To overcome signal overlapping, we developed a method designated as COmplementary Sequential assignment with MInimum Labeling Ensemble (COSMILE). According to this method, we generated three kinds of reverse-labeled samples to suppress signal overlapping. To assign the carbon signals sequentially, two-dimensional C^α(i+1)–C′C^α(i) correlation and dipolar assisted rotational resonance (DARR) experiments were performed under magic-angle sample spinning. On the basis of inter- and intra-residue ¹³C–¹³C chemical shift correlations, 97% of C^α, 97% of C^β and 92% of C′ signals were assigned directly from the spectra. Secondary structure analysis predicted a hairpin fold of two helices with a central loop. The effects of saturated and unsaturated phosphatidylcholines on TF_o c-ring structure were examined. The DARR spectra at 15 ms mixing time are essentially similar to each other in saturated and unsaturated lipid membranes, suggesting that TF_o c-rings have similar structures under the different environments. The spectrum of the sample in saturated lipid membranes showed better resolution and structural stability in the gel state. The C-terminal helix was suggested to locate in the outer layer of the c-ring.     

A c Subunit with Four Transmembrane Helices and One Ion (Na+)-binding Site in an Archaeal ATP Synthase: IMPLICATIONS FOR c RING FUNCTION AND STRUCTURE*

The ion-driven membrane rotors of ATP synthases consist of multiple copies of subunit c, forming a closed ring. Subunit c typically comprises two transmembrane helices, and the c ring features an ion-binding site in between each pair of adjacent subunits. Here, we use experimental and computational methods to study the structure and specificity of an archaeal c subunit more akin to those of V-type ATPases, namely that from Pyrococcus furiosus. The c subunit was purified by chloroform/methanol extraction and determined to be 15.8 kDa with four predicted transmembrane helices. However, labeling with DCCD as well as Na⁺-DCCD competition experiments revealed only one binding site for DCCD and Na⁺, indicating that the mature c subunit of this A₁A_O ATP synthase is indeed of the V-type. A structural model generated computationally revealed one Na⁺-binding site within each of the c subunits, mediated by a conserved glutamate side chain alongside other coordinating groups. An intriguing second glutamate located in-between adjacent c subunits was ruled out as a functional Na⁺-binding site. Molecular dynamics simulations indicate that the c ring of P. furiosus is highly Na⁺-specific under in vivo conditions, comparable with the Na⁺-dependent V₁V_O ATPase from Enterococcus hirae. Interestingly, the same holds true for the c ring from the methanogenic archaeon Methanobrevibacter ruminantium, whose c subunits also feature a V-type architecture but carry two Na⁺-binding sites instead. These findings are discussed in light of their physiological relevance and with respect to the mode of ion coupling in A₁A_O ATP synthases.     

Residues in the Polar Loop of Subunit c in Escherichia coli ATP Synthase Function in Gating Proton Transport to the Cytoplasm

Rotary catalysis in F₁F₀ ATP synthase is powered by proton translocation through the membrane-embedded F₀ sector. Proton binding and release occur in the middle of the membrane at Asp-61 on the second transmembrane helix (TMH) of subunit c, which folds in a hairpin-like structure with two TMHs. Previously, the aqueous accessibility of Cys substitutions in the transmembrane regions of subunit c was probed by testing the inhibitory effects of Ag⁺ or Cd²⁺ on function, which revealed extensive aqueous access in the region around Asp-61 and on the half of TMH2 extending to the cytoplasm. In the current study, we surveyed the Ag⁺ and Cd²⁺ sensitivity of Cys substitutions in the loop of the helical hairpin and used a variety of assays to categorize the mechanisms by which Ag⁺ or Cd²⁺ chelation with the Cys thiolates caused inhibition. We identified two distinct metal-sensitive regions in the cytoplasmic loop where function was inhibited by different mechanisms. Metal binding to Cys substitutions in the N-terminal half of the loop resulted in an uncoupling of F₁ from F₀ with release of F₁ from the membrane. In contrast, substitutions in the C-terminal half of the loop retained membrane-bound F₁ after metal treatment. In several of these cases, inhibition was shown to be due to blockage of passive H⁺ translocation through F₀ as assayed with F₀ reconstituted into liposomes. The results suggest that the C-terminal domain of the cytoplasmic loop may function in gating H⁺ translocation to the cytoplasm.     

Fluid Mechanical Matching of H-ATP Synthase Subunit c-Ring with Lipid Membranes Revealed by H Solid-State NMR

The F₁F_o-ATP synthase utilizes the transmembrane H⁺ gradient for the synthesis of ATP. F_o subunit c-ring plays a key role in transporting H⁺ through F_o in the membrane. We investigated the interactions of Escherichia coli subunit c with dimyristoylphosphatidylcholine (DMPC-d₅₄) at lipid/protein ratios of 50:1 and 20:1 by means of ²H-solid-state NMR. In the liquid-crystalline state of DMPC, the ²H-NMR moment values and the order parameter (S_CD) profile were little affected by the presence of subunit c, suggesting that the bilayer thickness in the liquid-crystalline state is matched to the transmembrane hydrophobic surface of subunit c. On the other hand, hydrophobic mismatch of subunit c with the lipid bilayer was observed in the gel state of DMPC. Moreover, the viscoelasticity represented by a square-law function of the ²H-NMR relaxation was also little influenced by subunit c in the fluid phase, in contrast with flexible nonionic detergents or rigid additives. Thus, the hydrophobic matching of the lipid bilayer to subunit c involves at least two factors, the hydrophobic length and the fluid mechanical property. These findings may be important for the torque generation in the rotary catalytic mechanism of the F₁F_o-ATPse molecular motor.     

Mussel and mammalian ATP synthase share the same bioenergetic cost of ATP

The molecular mechanism by which the membrane-embedded F_O sector of the mitochondrial ATP synthase translocates protons, thus dissipating the transmembrane protonmotive force and leading to ATP synthesis, involves the neutralization of the carboxylate residues of the c-ring. Carboxylates are thought to constitute the binding sites for ion translocation. In order to cast light on this mechanism, we exploited N,N’-dicyclohexylcarbodiimide, which covalently binds to F_O c-ring carboxylates, and ionophores which selectively modulate the transmembrane electric (Δφ) and chemical (ΔpH) gradients such as valinomycin, nigericin and dinitrophenol. ATP hydrolysis was evaluated in mitochondrial preparations and/or inside-out submitochondrial particles from mussel and mammalian tissues under different experimental conditions. The experiments pointed out striking similarities between mussel and mammalian mitochondrial ATP synthase. Our results support the hypothesis that the ATP synthase of Mytilus galloprovincialis induces intersubunit torque generation and translocates H⁺ by coordinating the hydronium ion (H₃O⁺) in the ion binding site of F_O. Our results are consistent with the hypothesis that in mussel mitochondria the main component of the electrochemical gradient driving proton flux and ATP synthesis is Δφ. Therefore, mussel F_O probably contains a small c-ring, which implies a low bioenergetic cost of making ATP as in mammals. These features which make mussel mitochondria as efficient in ATP production as mammalian ones may be especially advantageous in facultative aerobic species which intermittently exploit mitochondrial respiration to generate ATP.     

Fo-driven Rotation in the ATP Synthase Direction against the Force of F1 ATPase in the FoF1 ATP Synthase

Living organisms rely on the F_oF₁ ATP synthase to maintain the non-equilibrium chemical gradient of ATP to ADP and phosphate that provides the primary energy source for cellular processes. How the F_o motor uses a transmembrane electrochemical ion gradient to create clockwise torque that overcomes F₁ ATPase-driven counterclockwise torque at high ATP is a major unresolved question. Using single F_oF₁ molecules embedded in lipid bilayer nanodiscs, we now report the observation of F_o-dependent rotation of the c10 ring in the ATP synthase (clockwise) direction against the counterclockwise force of ATPase-driven rotation that occurs upon formation of a leash with F_o stator subunit a. Mutational studies indicate that the leash is important for ATP synthase activity and support a mechanism in which residues aGlu-196 and cArg-50 participate in the cytoplasmic proton half-channel to promote leash formation.