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.     

Functional and stoichiometric analysis of subunit e in bovine heart mitochondrial F<Subscript>0</Subscript>F<Subscript>1</Subscript>ATP synthase

The role of the integral inner membrane subunit e in self-association of F₀F₁ATP synthase from bovine heart mitochondria was analyzed by in situ limited proteolysis, blue native PAGE/iterative SDS-PAGE, and LC-MS/MS. Selective degradation of subunit e, without disrupting membrane integrity or ATPase capacity, altered the oligomeric distribution of F₀F₁ATP synthase, by eliminating oligomers and reducing dimers in favor of monomers. The stoichiometry of subunit e was determined by a quantitative MS-based proteomics approach, using synthetic isotope-labelled reference peptides IAQL*EEVK, VYGVGSL*ALYEK, and ELAEAQEDTIL*K to quantify the b, γ and e subunits, respectively. Accuracy of the method was demonstrated by confirming the 1:1 stoichiometry of subunits γ and b. Altogether, the results indicate that the integrity of a unique copy of subunit e is essential for self-association of mammalian F₀F₁ATP synthase. Elena Bisetto and Paola Picotti contributed equally to this work.     

Significance of the epsilon subunit in the thiol modulation of chloroplast ATP synthase

To understand the regulatory function of the gamma and epsilon subunits of chloroplast ATP synthase in the membrane integrated complex, we constructed a chimeric FoF1 complex of thermophilic bacteria. When a part of the chloroplast F1 gamma subunit was introduced into the bacterial FoF1 complex, the inverted membrane vesicles with this chimeric FoF1 did not exhibit the redox sensitive ATP hydrolysis activity, which is a common property of the chloroplast ATP synthase. However, when the whole part or the C-terminal alpha-helices region of the epsilon subunit was substituted with the corresponding region from CF1-epsilon together with the mutation of gamma, the redox regulation property emerged. In contrast, ATP synthesis activity did not become redox sensitive even if both the regulatory region of CF1-gamma and the entire epsilon subunit from CF1 were introduced. These results provide important features for the regulation of FoF1 by these subunits: (1) the interaction between gamma and epsilon is important for the redox regulation of FoF1 complex by the gamma subunit, and (2) a certain structural matching between these regulatory subunits and the catalytic core of the enzyme must be required to confer the complete redox regulation mechanism to the bacterial FoF1. In addition, a structural requirement for the redox regulation of ATP hydrolysis activity might be different from that for the ATP synthesis activity.     

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.     

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.     

Subunit Organization of the Stator Part of the F 0 Complex from Escherichia coli ATP Synthase

Membrane-bound ATP synthases (F₁F₀) catalyze the synthesis of ATP via a rotary catalyticmechanism utilizing the energy of an electrochemical ion gradient. The transmembrane potentialis supposed to propel rotation of a subunit c ring of F₀ together with subunits and of F₁,hereby forming the rotor part of the enzyme, whereas the remainder of the F₁F₀ complexfunctions as a stator for compensation of the torque generated during rotation. This reviewfocuses on our recent work on the stator part of the F₀ complex, e.g., subunits a and b. Usingepitope insertion and antibody binding, subunit a was shown to comprise six transmembranehelixes with both the N- and C-terminus oriented toward the cytoplasm. By use of circulardichroism (CD) spectroscopy, the secondary structure of subunit b incorporated intoproteoliposomes was determined to be 80% -helical together with 14% turn conformation, providingflexibility to the second stalk. Reconstituted subunit b together with isolated ac subcomplexwas shown to be active in proton translocation and functional F₁ binding revealing the nativeconformation of the polypeptide chain. Chemical crosslinking in everted membrane vesiclesled to the formation of subunit b homodimers around residues bQ37 to bL65, whereas bA32Ccould be crosslinked to subunit a, indicating a close proximity of subunits a and b near themembrane. Further evidence for the proposed direct interaction between subunits a and b wasobtained by purification of a stable ab ₂ subcomplex via affinity chromatography using Histags fused to subunit a or b. This ab ₂ subcomplex was shown to be active in proton translocationand F₁ binding, when coreconstituted with subunit c. Consequences of crosslink formationand subunit interaction within the F₁F₀ complex are discussed.     

Met23Lys mutation in subunit gamma of FOF1-ATP synthase from Rhodobacter capsulatus impairs the activation of ATP hydrolysis by protonmotive force

H⁺-F_OF₁-ATP synthase couples proton flow through its membrane portion, F_O, to the synthesis of ATP in its headpiece, F₁. Upon reversal of the reaction the enzyme functions as a proton pumping ATPase. Even in the simplest bacterial enzyme the ATPase activity is regulated by several mechanisms, involving inhibition by MgADP, conformational transitions of the ε subunit, and activation by protonmotive force. Here we report that the Met23Lys mutation in the γ subunit of the Rhodobacter capsulatus ATP synthase significantly impaired the activation of ATP hydrolysis by protonmotive force. The impairment in the mutant was due to faster enzyme deactivation that was particularly evident at low ATP/ADP ratio. We suggest that the electrostatic interaction of the introduced γLys23 with the DELSEED region of subunit β stabilized the ADP-inhibited state of the enzyme by hindering the rotation of subunit γ rotation which is necessary for the activation.     

The <Emphasis Type="Italic">b</Emphasis><Subscript>arg36</Subscript> contributes to efficient coupling in F<Subscript>1</Subscript>F<Subscript>O</Subscript> ATP synthase in <Emphasis Type="Italic">Escherichia coli</Emphasis>

In Escherichia coli, the F₁F_O ATP synthase b subunits house a conserved arginine in the tether domain at position 36 where the subunit emerges from the membrane. Previous experiments showed that substitution of isoleucine or glutamate result in a loss of enzyme activity. Double mutants have been constructed in an attempt to achieve an intragenic suppressor of the b _arg36→ile and the b _arg36→glu mutations. The b _arg36→ile mutation could not be suppressed. In contrast, the phenotypic defect resulting from the b _arg36→glu mutation was largely suppressed in the b _{arg36→glu,glu39→arg} double mutant. E. coli expressing the b _{arg36→glu,glu39→arg} subunit grew well on succinate-based medium. F₁F_O ATP synthase complexes were more efficiently assembled and ATP driven proton pumping activity was improved. The evidence suggests that efficient coupling in F₁F_O ATP synthase is dependent upon a basic amino acid located at the base of the peripheral stalk.     

ADP and ATP binding to noncatalytic sites of thiol-modulated chloroplast ATP synthase

A modified ‘cold chase’ technique was used to study tight [¹⁴C]ADP and [¹⁴C]ATP binding to noncatalytic sites of chloroplast ATP synthase (CF₀F₁). The binding was very low in the dark and sharply increased with light intensity. Dissociation of labeled nucleotides incorporated into noncatalytic sites of CF₀F₁ or CF₁ reconstituted with EDTA-treated thylakoid membranes was also found to be light-dependent. Time dependence of nucleotide dissociation is described by the first order equation with a k _d of about 5 min⁻¹. The exposure of thylakoid membranes to 0.7–24.8 μM nucleotides leads to filling of up to two noncatalytic sites of CF₀F₁. The sites differ in their specificity: one preferentially binds ADP, whereas the other – ATP. A much higher ATP/ADP ratio of nucleotides bound at noncatalytic sites of isolated CF₁ dramatically decreases upon its reconstitution with EDTA-treated thylakoid membranes. It is suggested that the decrease is caused by conformational changes in one of the α subunits induced by its interaction with the δ subunit and/or subunit I–II when CF₁ becomes bound to a thylakoid membrane.     

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.     

Subunit rotation in F0F1-ATP synthases as a means of coupling proton transport through F0 to the binding changes in F1

The rotation of an asymmetric core of subunits in F₀F₁-ATP synthases has been proposed as a means of coupling the exergonic transport of protons through F₀ to the endergonic conformational changes in F₁ required for substrate binding and product release. Here we review earlier evidence both for and against subunit rotation and then discuss our most recent studies using reversible intersubunit disulfide cross-links to test for rotation. We conclude that the subunit of F₁ rotates relative to the surrounding catalytic subunits during catalytic turnover by both soluble F₁ and membrane-bound F₀F₁. Furthermore, the inhibition of this rotation by the modification of F₀ with DCCD suggests that rotation in F₁ is obligatorily coupled to rotation in F₀ as an integral part of the coupling mechanism.     

ATP Synthases With Novel Rotor Subunits: New Insights into Structure,Function and Evolution of ATPases

ATPases with unusual membrane-embedded rotor subunits were found in both F₁F₀ and A₁A₀ ATP synthases. The rotor subunit c of A₁A₀ ATPases is, in most cases, similar to subunit c from F₀. Surprisingly, multiplied c subunits with four, six, or even 26 transmembrane spans have been found in some archaea and these multiplication events were sometimes accompanied by loss of the ion-translocating group. Nevertheless, these enzymes are still active as ATP synthases. A duplicated c subunit with only one ion-translocating group was found along with “normal” F₀ c subunits in the Na⁺ F₁F₀ ATP synthase of the bacterium Acetobacterium woodii. These extraordinary features and exceptional structural and functional variability in the rotor of ATP synthases may have arisen as an adaptation to different cellular needs and the extreme physicochemical conditions in the early history of life.     

Mutagenesis Studies of the F1F0 ATP Synthase b Subunit Membrane Domain

A homodimer of b subunits constitutes the peripheral stalk linking the F₁ and F₀ sectors of the Escherichia coli ATP synthase. Each b subunit has a single-membrane domain. The constraints on the membrane domain have been studied by systematic mutagenesis. Replacement of a segment proximal to the cytoplasmic side of the membrane had minimal impact on F₁F₀ ATP synthase. However, multiple substitutions on the periplasmic side resulted in defects in assembly of the enzyme complex. These mutants had insufficient oxidative phosphorylation to support growth, and biochemical studies showed little F₁F₀ ATPase and no detectable ATP-driven proton pumping activity. Expression of the b _N2A,T6A,Q10A subunit was also oxidative phosphorylation deficient, but the b _N2A,T6A,Q10A protein was incorporated into an F₁F₀ complex. Single amino acid substitutions had minimal reductions in F₁F₀ ATP synthase function. The evidence suggests that the b subunit membrane domain has several sites of interaction contributing to assembly of F₀, and that these interactions are strongest on the periplasmic side of the bilayer.     

Essential arginine in subunit a and aspartate in subunit c of FoF1 ATP synthase: Effect of repositioning within Helix 4 of subunit a and Helix 2 of subunit c

F_oF₁ ATP synthase couples proton flow through the integral membrane portion F_o (ab₂c₁₀) to ATP-synthesis in the extrinsic F₁-part ((αβ)₃γδε) (Escherichia coli nomenclature and stoichiometry). Coupling occurs by mechanical rotation of subunits c₁₀γε relative to (αβ)₃δab₂. Two residues were found to be essential for proton flow through ab₂c_10, namely Arg₂₁₀ in subunit a (aR210) and Asp₆₁ in subunits c (cD61). Their deletion abolishes proton flow, but “horizontal” repositioning, by anchoring them in adjacent transmembrane helices, restores function. Here, we investigated the effects of “vertical” repositioning aR210, cD61, or both by one helical turn towards the N- or C-termini of their original helices. Other than in the horizontal the vertical displacement changes the positions of the side chains within the depth of the membrane. Mutant aR210A/aN214R appeared to be short-circuited in that it supported proton conduction only through EF₁-depleted EF_o, but not in EF_oEF_1, nor ATP-driven proton pumping. Mutant cD61N/cM65D grew on succinate, retained the ability to synthesize ATP and supported passive proton conduction but apparently not ATP hydrolysis-driven proton pumping.     

Osmomechanics of the Propionigenium modestum Fo Motor

In Propionigenium modestum, ATP is manufactured from ADP and phosphate by the enzyme ATP synthase using the free energy of an electrochemical gradient of Na⁺ ions. The P. modestum ATP synthase is a clear member of the family of F-type ATP synthases and the only major distinction is an extension of the coupling ion specificity to H⁺, Li⁺, or Na⁺, depending on the conditions. The use of Na⁺ as a coupling ion offers unique experimental options to decipher the ion-translocation mechanism and the osmotic and mechanical behavior of the enzyme. The single a subunit and the oligomer of c subunits are part of the stator and rotor, respectively, and operate together in the ion-translocation mechanism. During ATP synthesis, Na⁺ diffuses from the periplasm through the a subunit channel onto the Na⁺ binding site on a c subunit. From there it dissociates into the cytoplasm after the site has rotated out of the interface with subunit a. In the absence of a membrane potential, the rotor performs Brownian motions into either direction and Na⁺ ions are exchanged between the two compartments separated by the membrane. Upon applying voltage, however, the direction of Na⁺ flux and of rotation is biased by the potential. The motor generates torque to drive the rotation of the subunit, thereby releasing tightly bound ATP from catalytic sites in F₁. Hence, the membrane potential plays a pivotal role in the torque-generating mechanism. This is corroborated by the fact that for ATP synthesis, at physiological rates, the membrane potential is indispensable. We propose a catalytic mechanism for torque generation by the F_o motor that is in accord with all experimental data and is in quantitative agreement with the requirement for ATP synthesis.     

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.     

ATP Synthases in the Year 2000: Defining the Different Levels of Mechanism and Getting a Grip on Each

ATP synthases are unusually complex molecules, which fractionate most readily into two major units, one a water soluble unit called F₁ and the other a detergent soluble unit called F₀. In almost all known species the F₁ unit consists of 5 subunit types in the stoichiometric ratio ₃₃ while the F₀ unit contains 3 subunit types (a, b, and c) in E. coli, and at least 10 subunit types (a, b, c, and others) in higher animals. It is now believed by many investigators that during the synthesis of ATP, protons derived from an electrochemical gradient generated by an electron transport chain are directed through the F₀ unit in such a way as to drive the rotation of the single subunit, which extends from an oligomeric ring of at least 10 c subunits in F₀ through the center of F₁. It is further believed by many that the rotating subunit, by interacting sequentially with the 3 pairs of F₁ (360° cycle) in the presence of ADP, P_i, and Mg⁺⁺, brings about via power strokes conformational/binding changes in these subunits that promote the synthesis of ATP and its release on each pair. In support of these views, studies in several laboratories either suggest or demonstrate that F₀ consists in part of a proton gradient driven motor while F₁ consists of an ATP hydrolysis driven motor, and that the subunit does rotate during F₁ function. Therefore, current implications are that during ATP synthesis the former motor drives the latter in reverse via the subunit. This would suggest that the process of understanding the mechanism of ATP synthases can be subdivided into three major levels, which include elucidating those chemical and/or biophysical events involved in (1) inducing rotation of the subunit, (2) coupling rotation of this subunit to conformational/binding changes in each of the 3 pairs, and (3) forming ATP and water (from ADP, P_i, and Mg⁺⁺) and then releasing these products from each of the 3 catalytic sites. Significantly, it is at the final level of mechanism where the bond breaking/making events of ATP synthesis occur in the transition state, with the former two levels of mechanism setting the stage for this critical payoff event. Nevertheless, in order to get a better grip in this new century on how ATP synthases make ATP and then release it, we must take on the difficult challenge of elucidating each of the three levels of mechanism.     

Structure of theEscherichia coli ATP synthase and role of the γ and ε subunits in coupling catalytic site and proton channeling functions

The structure of theEscherichia coli ATP synthase has been studied by electron microscopy and a model developed in which the and subunits of the F₁ part are arranged hexagonally (in top view) alternating with one another and surrounding a central cavity of around 35 Å at its widest point. The and subunits are interdigitated in side view for around 60 Å of the 90 Å length of the molecule. The F₁ narrows and has three-fold symmetry at the end furthest from the F₀ part. The F₁ is linked to F₀ by a stalk approximately 45 Å long and 25–30 Å in diameter. The F₀ part is mostly buried in the lipid bilayer. The subunit provides a domain that extends into the central cavity of the F₁ part. The and subunits are in a different conformation when ATP+Mg²⁺ are present in catalytic sites than when ATP+EDTA are present. This is consistent with these two small subunits switching conformations as a function of whether or not phosphate is bound to the enzyme at the position of the phosphate of ATP. We suggest that this switching is the key to the coupling of catalytic site events with proton translocation in the F₀ part of the complex.     

The molecular mechanism of ATP synthesis by F1F0-ATP synthase

ATP synthesis by oxidative phosphorylation and photophosphorylation, catalyzed by F₁F₀-ATP synthase, is the fundamental means of cell energy production. Earlier mutagenesis studies had gone some way to describing the mechanism. More recently, several X-ray structures at atomic resolution have pictured the catalytic sites, and real-time video recordings of subunit rotation have left no doubt of the nature of energy coupling between the transmembrane proton gradient and the catalytic sites in this extraordinary molecular motor. Nonetheless, the molecular events that are required to accomplish the chemical synthesis of ATP remain undefined. In this review we summarize current state of knowledge and present a hypothesis for the molecular mechanism of ATP synthesis.     

Regulation of Proton Flow and ATP Synthesis in Chloroplasts

The chloroplast ATP synthase is strictly regulated so that it is very active in the light (rates of ATP synthesis can be higher than 5 mol/min/mg protein), but virtually inactive in the dark. The subunits of the catalytic portion of the ATP synthase involved in activation, as well as the effects of nucleotides are discussed. The relation of activation to proton flux through the ATP synthase and to changes in the structure of enzyme induced by the proton electrochemical gradient are also presented. It is concluded that the and subunits of CF₁ play key roles in both regulation of activity and proton translocation.