The mechanism of rotating proton pumping ATPases

Two proton pumps, the F-ATPase (ATP synthase, F_oF₁) and the V-ATPase (endomembrane proton pump), have different physiological functions, but are similar in subunit structure and mechanism. They are composed of a membrane extrinsic (F₁ or V₁) and a membrane intrinsic (F_o or V_o) sector, and couple catalysis of ATP synthesis or hydrolysis to proton transport by a rotational mechanism. The mechanism of rotation has been extensively studied by kinetic, thermodynamic and physiological approaches. Techniques for observing subunit rotation have been developed. Observations of micron-length actin filaments, or polystyrene or gold beads attached to rotor subunits have been highly informative of the rotational behavior of ATP hydrolysis-driven rotation. Single molecule FRET experiments between fluorescent probes attached to rotor and stator subunits have been used effectively in monitoring proton motive force-driven rotation in the ATP synthesis reaction. By using small gold beads with diameters of 40-60 nm, the E. coli F₁ sector was found to rotate at surprisingly high speeds (> 400 rps). This experimental system was used to assess the kinetics and thermodynamics of mutant enzymes. The results revealed that the enzymatic reaction steps and the timing of the domain interactions among the β subunits, or between the β and γ subunits, are coordinated in a manner that lowers the activation energy for all steps and avoids deep energy wells through the rotationally-coupled steady-state reaction. In this review, we focus on the mechanism of steady-state F₁-ATPase rotation, which maximizes the coupling efficiency between catalysis and rotation.     

H+ transport and coupling by the F0 sector of the ATP synthase: Insights into the molecular mechanism of function

The F₀ sector of the ATP synthase complex facilitates proton translocation through the membrane, and via interaction with the F₁ sector, couples proton transport to ATP synthesis. The molecular mechanism of function is being probed by a combination of mutant analysis and structural biochemistry, and recent progress on theEscherichia coli F₀ sector is reviewed here. TheE. coli F₀ is composed of three types of subunits (a, b, andc) and current information on their folding and organization in F₀ is reviewed. The structure of purified subunitc in chloroform-methanol-H₂O resembles that in native F₀, and progress in determining the structure by NMR methods is reviewed. Genetic experiments suggest that the two helices of subunitc must interact as a functional unit around an essential carboxyl group as protons are transported. In addition, a unique class of suppressor mutations identify a transmembrane helix of subunita that is proposed to interact with the bihelical unit of subunitc during proton transport. The role of multiple units of subunitc in coupling proton translocation to ATP synthesis is considered. The special roles of Asp61 of subunitc and Arg210 of subunita in proton translocation are also discussed.     

Membrane tubulation and proton pumps

Summary A model is presented for one type of membrane tubulation. In this model large transmembrane protein complexes link together to form helical bands. To form the helical bands the individual complexes within the band would need to form a slight bend and twist with adjacent complexes as they link together. Such linkages would lead to the band and lipid bilayer extending out from the plane of the membrane to form a tube of a diameter equal to or smaller than the radial diameter of the helix. This model is supported by two examples of membrane tubulation found inParamecium, in which proton pumps are the transmembrane complexes that are associated together to form the helically coiled bands. These include the helically linked F₀F₁ complexes of the mitochondrial inner membrane and the V₀V₁ complexes of the decorated tubules of the contractile vacuole complex. Such tubules result in very high surface-to-volume ratios and may be important in efficient ATP production or in forming proton gradients for transporting ions across membranes.     

The coupling of the relative movement of thea andc subunits of the F0 to the conformational changes in the F1-ATPase

F₀F₁-ATPase structural information gained from X-ray crystallography and electron microscopy has activated interest in a rotational mechanism for the F₀F₁-ATPase. Because of the subunit stoichiometry and the involvement of both thea- andc-subunits in the mechanism of proton movement, it is argued that relative movement must occur between the subunits. Various options for the arrangement and structure of the subunits involved are discussed and a mechanism proposed.     

Cardiolipin is essential for higher proton translocation activity of reconstituted F0

The F_o membrane domain of F_oF₁-ATPase complex had been purified from porcine heart mitochondria. SDS-PAGE with silver staining indicated that the purity of F_o was about 85% and the sample contained no subunits of F₁-ATPase. The purified F_o was reconstituted into liposomes with different phospholipid composition, and the effect of CL (cardiolipin), PA (phosphatidic acid), PI (phosphatidylinositol) and PS (phosphatidylserine) on the H⁺ translocation activity of F_o was investigated. The results demonstrated that CL, PA and PI could promote the proton translocation of F_o with the order of CL＞PA＞＞PI, while PS inhibited it. Meanwhile ADM (adriamycin) severely impaired the proton translocation activity of F_o vesicles containing CL, which suggested that CL’s stimulation of the activity of reconstituted F_o might correlate with its non-bilayer propensity. After F_o was incorporated into the liposomes containing PE (phosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine) as well as DEPE (dielaidoylphosphatidylethanolamine), it was found that the proton translocation activity of F_o vesicles increased with the increasing content of PE or DOPE, which has high propensity of forming non-bilayer structure, but was independent of DEPE. The dynamic quenching of the intrinsic fluorescence of tryptophan by HB (hypocrellin B) as well as fluorescent spectrum of acrylodan labeling F_o at cysteine indicated that CL could induce F_o to a suitable conformation resulting in higher proton translocation activity.     

Cardiolipin is essential for higher proton translocation activity of reconstituted F0

The F₀ membrane domain of F₀F₁-ATPase complex had been purified from porcine heart mitochondria. SDS-PAGE with silver staining indicated that the purity of F₀ was about 85% and the sample contained no subunits of F₁-ATPase. The purified F₀ was reconstituted into liposomes with different phospholipid composition, and the effect of CL (cardiolipin), PA (phosphatidic acid), PI (phosphatidylinositol) and PS (phosphatidylserine) on the H⁺ translocation activity of F₀ was investigated. The results demonstrated that CL, PA and PI could promote the proton translocation of F₀ with the order of CLPA>PI, while PS inhibited it. Meanwhile ADM (adriamycin) severely impaired the proton translocation activity of F₀ vesicles containing CL, which suggested that CL’s stimulation of the activity of reconstituted F₀ might correlate with its non-bilayer propensity. After F₀ was incorporated into the liposomes containing PE (phosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine) as well as DEPE (dielaidoylphosphatidylethanolamine), it was found that the proton translocation activity of F₀ vesicles increased with the increasing content of PE or DOPE, which has high propensity of forming non-bilayer structure, but was independent of DEPE. The dynamic quenching of the intrinsic fluorescence of tryptophan by HB (hypocrellin B) as well as fluorescent spectrum of acrylodan labeling F₀ at cysteine indicated that CL could induce F₀ to a suitable conformation resulting in higher proton translocation activity.     

Relevance of Divalent Cations to ATP-Driven Proton Pumping in Beef Heart Mitochondrial F0F1-ATPase

The ATP hydrolysis rate and the ATP hydrolysis-linked proton translocation by the F₀F₁-ATPase of beef heart submitochondrial particles were examined in the presence of several divalent metal cations. All Me–ATP complexes tested sustained ATP hydrolysis, although to a different extent. However, only Mg- and Mn-ATP-dependent hydrolysis could sustain a high level of proton pumping activity, as determined by acridine fluorescence quenching. Moreover, the K _m of the Me-ATP hydrolysis-induced proton pumping activity was very similar to the K _m value of Me-ATP hydrolysis. Both oligomycin and DCCD caused the full recovery of the fluorescence, providing clear evidence for the association of Mg-ATP hydrolysis with proton translocation through the F₀F₁-ATPase complex. In contrast, with other Me-ATP complexes, including Ca-ATP as substrate, the proton pumping activity was undetectable, implicating an uncoupling nature for these substrates. Attempts to demonstrate the involvement of the subunit of the enzyme in the coupling mechanism failed, suggesting that the participation of at least the N-terminal segment of the subunit in the coupling mechanism of the mitochondrial enzyme is unlikely.     

Assembly of the Escherichia coli F1F0 ATPase, a large multimeric membrane-bound enzyme

The F₁F₀ proton translocating ATPase of Escherichia coli is a large membrane-bound enzyme complex consisting of more than 20 polypeptides that are encoded by the unc operon. Besides being a system for analysing the enzymology of ATP synthesis and energy coupling, the ATPase is a model system for determining how large oligomeric membrane-bound proteins are synthesized and assembled. The assembly of the ATPase involves differential gene expression and assembly of the subunits within the membrane and with each other. This review discusses the influence of F₁ subunits on the assembly and proton permeability of the F₀ proton channel, and the possible advantages to assembly of the particular arrangement of genes in the unc operon.     

ATP synthase: Subunit–subunit interactions in the stator stalk

In ATP synthase, proton translocation through the F_o subcomplex and ATP synthesis/hydrolysis in the F₁ subcomplex are coupled by subunit rotation. The static, non-rotating portions of F₁ and F_o are attached to each other via the peripheral “stator stalk”, which has to withstand elastic strain during subunit rotation. In Escherichia coli, the stator stalk consists of subunits b₂δ; in other organisms, it has three or four different subunits. Recent advances in this area include affinity measurements between individual components of the stator stalk as well as a detailed analysis of the interaction between subunit δ (or its mitochondrial counterpart, the oligomycin-sensitivity conferring protein, OSCP) and F₁. The current status of our knowledge of the structure of the stator stalk and of the interactions between its subunits will be discussed in this review.     

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.     

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.     

Direct observation of stepped proteolipid ring rotation in E. coli F₀F₁-ATP synthase

Although single‐molecule experiments have provided mechanistic insight for several molecular motors, these approaches have proved difficult for membrane bound molecular motors like the F_oF₁‐ATP synthase, in which proton transport across a membrane is used to synthesize ATP. Resolution of smaller steps in F_o has been particularly hampered by signal‐to‐noise and time resolution. Here, we show the presence of a transient dwell between F_o subunits a and c by improving the time resolution to 10 μs at unprecedented S/N, and by using Escherichia coli F_oF₁ embedded in lipid bilayer nanodiscs. The transient dwell interaction requires 163 μs to form and 175 μs to dissociate, is independent of proton transport residues aR210 and cD61, and behaves as a leash that allows rotary motion of the c‐ring to a limit of ～36° while engaged. This leash behaviour satisfies a requirement of a Brownian ratchet mechanism for the F_o motor where c‐ring rotational diffusion is limited to 36°.     

Sodium-translocating adenosine triphosphatase inStreptococcus faecalis

Sodium-transloating ATPase in the fermentative bacteriumStreptococcus faecalis exchanges sodium for potassium ions. Sodium ions stimulate its activity, but K⁺ ions have no significant effect at present. Although the molecular nature of the sodium ATPase is not clear, the enzyme is distinct from other ion-motive ATPases (E₁E₂ type and F₁F₀ type) as judged by its resistance to vanadate as well as dicyclohexylcarbodiimde. The sodium ATPase is induced when cells are grown on media rich in sodium, particularly under conditions that limit the generation of a proton potential or block the constitutive sodium/proton antiporter, indicating that an increase in the cytoplasmic sodium level serves as the signal. The enzyme is not induced in response to K⁺ deprivation. The sodium ATPase may have evolved to cope with a sodium-rich environment under conditions that limit the magnitude of the proton potential.     

The proton-driven rotor of ATP synthase: ohmic conductance (10 fS), and absence of voltage gating

The membrane portion of F₀F₁-ATP synthase, F₀, translocates protons by a rotary mechanism. Proton conduction by F₀ was studied in chromatophores of the photosynthetic bacterium Rhodobacter capsulatus. The discharge of a light-induced voltage jump was monitored by electrochromic absorption transients to yield the unitary conductance of F₀. The current-voltage relationship of F₀ was linear from 7 to 70 mV. The current was extremely proton-specific (>10⁷) and varied only slightly (≈threefold) from pH 6 to 10. The maximum conductance was ≈10 fS at pH 8, equivalent to 6240 H⁺ s⁻¹ at 100-mV driving force, which is an order-of-magnitude greater than of coupled F₀F₁. There was no voltage-gating of F₀ even at low voltage, and proton translocation could be driven by ΔpH alone, without voltage. The reported voltage gating in F₀F₁ is thus attributable to the interaction of F₀ with F₁ but not to F₀ proper. We simulated proton conduction by a minimal rotary model including the rotating c-ring and two relay groups mediating proton exchange between the ring and the respective membrane surface. The data fit attributed pK values of ≈6 and ≈10 to these relays, and placed them close to the membrane/electrolyte interface.     

A Biological Molecular Motor, Proton-Translocating ATP Synthase: Multidisciplinary Approach for a Unique Membrane Enzyme

Proton-translocating ATP synthase (F_oF₁) synthesizes ATP from ADP and phosphate, coupled with an electrochemical proton gradient across the biological membrane. It has been established that the rotation of a subunit assembly is an essential feature of the enzyme mechanism and that F_oF₁ can be regarded as a molecular motor. Thus, experimentally, in the reverse direction (ATP hydrolysis), the chemical reaction drives the rotation of a c _10-14 subunit assembly followed by proton translocation. We discuss our very recent results regarding subunit rotation in Escherichia coli F_oF₁ with a combined biophysical and mutational approach.     

Resolving the Negative Potential Side (n-side) Water-accessible Proton Pathway of F-type ATP Synthase by Molecular Dynamics Simulations

The rotation of F₁F_o-ATP synthase is powered by the proton motive force across the energy-transducing membrane. The protein complex functions like a turbine; the proton flow drives the rotation of the c-ring of the transmembrane F_o domain, which is coupled to the ATP-producing F₁ domain. The hairpin-structured c-protomers transport the protons by reversible protonation/deprotonation of a conserved Asp/Glu at the outer transmembrane helix (TMH). An open question is the proton transfer pathway through the membrane at atomic resolution. The protons are thought to be transferred via two half-channels to and from the conserved cAsp/Glu in the middle of the membrane. By molecular dynamics simulations of c-ring structures in a lipid bilayer, we mapped a water channel as one of the half-channels. We also analyzed the suppressor mutant cP24D/E61G in which the functional carboxylate is shifted to the inner TMH of the c-protomers. Current models concentrating on the “locked” and “open” conformations of the conserved carboxylate side chain are unable to explain the molecular function of this mutant. Our molecular dynamics simulations revealed an extended water channel with additional water molecules bridging the distance of the outer to the inner TMH. We suggest that the geometry of the water channel is an important feature for the molecular function of the membrane part of F₁F_o-ATP synthase. The inclination of the proton pathway isolates the two half-channels and may contribute to a favorable clockwise rotation in ATP synthesis mode.     

Control of rotation of the F<Subscript>1</Subscript>F<Subscript>O</Subscript>-ATP synthase nanomotor by an inhibitory α-helix from unfolded ε or intrinsically disordered ζ and IF<Subscript>1</Subscript> proteins

The ATP synthase is a ubiquitous nanomotor that fuels life by the synthesis of the chemical energy of ATP. In order to synthesize ATP, this enzyme is capable of rotating its central rotor in a reversible manner. In the clockwise (CW) direction, it functions as ATP synthase, while in counter clockwise (CCW) sense it functions as an proton pumping ATPase. In bacteria and mitochondria, there are two known canonical natural inhibitor proteins, namely the ε and IF₁ subunits. These proteins regulate the CCW F₁F_O-ATPase activity by blocking γ subunit rotation at the α_DP/β_DP/γ subunit interface in the F₁ domain. Recently, we discovered a unique natural F₁-ATPase inhibitor in Paracoccus denitrificans and related α-proteobacteria denoted the ζ subunit. Here, we compare the functional and structural mechanisms of ε, IF₁, and ζ, and using the current data in the field, it is evident that all three regulatory proteins interact with the α_DP/β_DP/γ interface of the F₁-ATPase. In order to exert inhibition, IF₁ and ζ contain an intrinsically disordered N-terminal protein region (IDPr) that folds into an α-helix when inserted in the α_DP/β_DP/γ interface. In this context, we revised here the mechanism and role of the ζ subunit as a unidirectional F-ATPase inhibitor blocking exclusively the CCW F₁F_O-ATPase rotation, without affecting the CW-F₁F_O-ATP synthase turnover. In summary, the ζ subunit has a mode of action similar to mitochondrial IF₁, but in α-proteobacteria. The structural and functional implications of these intrinsically disordered ζ and IF₁ inhibitors are discussed to shed light on the control mechanisms of the ATP synthase nanomotor from an evolutionary perspective.     

Partial Assembly of the Yeast Mitochondrial ATP Synthase 1

The mitochondrial ATP synthase is a molecular motor that drives the phosphorylation ofADP to ATP. The yeast mitochondrial ATP synthase is composed of at least 19 differentpeptides, which comprise the F₁ catalytic domain, the F₀ proton pore, and two stalks, oneof which is thought to act as a stator to link and hold F₁ to F₀, and the other as a rotor.Genetic studies using yeast Saccharomyces cerevisiae have suggested the hypothesis thatthe yeast mitochondrial ATP synthase can be assembled in the absence of 1, and even 2, ofthe polypeptides that are thought to comprise the rotor. However, the enzyme complexassembled in the absence of the rotor is thought to be uncoupled, allowing protons to freelyflow through F₀ into the mitochondrial matrix. Left uncontrolled, this is a lethal process andthe cell must eliminate this leak if it is to survive. In yeast, the cell is thought to lose ordelete its mitochondrial DNA (the petite mutation) thereby eliminating the genes encodingessential components of F₀. Recent biochemical studies in yeast, and prior studies in E. coli,have provided support for the assembly of a partial ATP synthase in which the ATP synthaseis no longer coupled to proton translocation.     

Properties and new methods of non-equilibrium membrane bound proton fraction research under conditions of proton pump activation

Under the conditions of low-amplitude mitochondrial swelling, the oxidative phosphorylation system functions in a local coupling mode postulated by Williams in 1961. The proton pumps activation leads to the formation of non-equilibrium membrane bounded proton fraction (nef-H⁺), which is sorbed on the outer side of the inner mitochondrial membrane under these conditions. This proton fraction is crucial for the ATP synthesis. The present work is devoted to the development of the methods allowing investigations of the properties of nef-H⁺. For this purpose, a new membranotropic highly hydrophobic uncoupler 2,4,6-trichloro-3-pentadecylphenol was synthesized. In accordance with our results, it can be referred to as a new type of transmemebrane proton carriers, which interact specifically only with nef-H⁺ outer side of the inner membrane. A new method of the nef-H⁺ removal from the outer side of the inner mitochondrial membrane under the conditions when the proton pumps are active has been developed. The method is based on neutralization of nef-H⁺ by hydroxyl anions transferred by H₂PO₄⁻/OH⁻-antiporter to interfacial border. It was demonstrated that the membrane proton fraction is heterogeneous. Two components of the fraction were identified. And at the same time, it was shown that nucleotide translocator participates in the formation of one component. Also, in this series of experiments it was found that a well-known effect of respiration inhibition by high concentrations of uncoupler under certain conditions can be completely explained by the nef-H⁺ formation. We presume that one of the most important results of the studies carried out is a new independent evidence of the existence of nef-H⁺, which is formed under the conditions of proton pump activation.