Coupling Proton Movement to ATP Synthesis in the Chloroplast ATP Synthase

The chloroplast F₀F₁-ATP synthase-ATPase is a tiny rotary motor responsible for coupling ATP synthesis and hydrolysis to the light-driven electrochemical proton gradient. Reversible oxidation/reduction of a dithiol, located within a special regulatory domain of the γ subunit of the chloroplast F₁ enzyme, switches the enzyme between an inactive and an active state. This regulatory mechanism is unique to the ATP synthases of higher plants and its physiological significance lies in preventing nonproductive depletion of essential ATP pools in the dark. The three-dimensional structure of the chloroplast F₁ gamma subunit has not yet been solved. To examine the mechanism of dithiol regulation, a model of the chloroplast gamma subunit was obtained through segmental homology modeling based on the known structures of the mitochondrial and bacterial γ subunits, together with de novo construction of the unknown regulatory domain. The model has provided considerable insight into how the dithiol might modulate catalytic function. This has, in turn, suggested a mechanism by which rotation of subunits in F₀, the transmembrane proton channel portion of the enzyme, can be coupled, via the ε subunit, to rotation of the γ subunit of F₁ to achieve the 120° (or 90°+30°) stepping action that is characteristic of F₁ γ subunit rotation.     

The Saccharomyces cerevisiae ATP Synthase

The ATP synthase of the yeast Saccharomyces cerevisiae is composed of 20 different subunitswhose primary structure is known. The organization of proteins that constitute the membranousdomain is now under investigation. Cysteine insertions combined with the use of nonpermeantmaleimide reagents and cross-linking reagents showing different lengths and specificitycontribute to the knowledge of the location of the N- and C-termini of the subunits involved in thestator of the enzyme and their organization. This review summarizes data on yeast ATP synthaseobtained in our laboratory since 1980.     

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

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

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.     

Chemomechanical Coupling in Single-Molecule F-Type ATP Synthase

An extremely small reaction chamber with a volume of a few femtoliters was developed for a highly sensitive detection of biological reaction. By encapsulating a single F(1)-ATPase (F(1)) molecule with ADP and an inorganic phosphate in the chamber, the chemomechanical coupling efficiency of ATP synthesis catalyzed by reversely rotated F(1) was successfully determined (Rondelez et al., 2005a, Nature, 444, 773-777). While the alpha3beta3gamma subcomplex of F(1) generated ATP with a low efficiency (approximately 10%), inclusion of the epsilon subunit into the subcomplex enhanced the efficiency up to 77%. This raises a new question about the mechanism of F(0)F(1)-ATP synthase (F(0)F(1)): How does the epsilon subunit support the highly coupled ATP synthesis of F(1)? To address this question, we measured the conformational dynamics of the epsilon subunit using fluorescence resonance energy transfer (FRET) at the single-molecule level. The experimental data revealed epsilon changes the conformation of its C-terminus helices in a nucleotide-dependent manner. It is plausible that the conformational change of epsilon switches the catalytic mode of F(0)F(1) for highly coupled ATP synthesis.     

Role of alphaPhe-291 residue in the phosphate-binding subdomain of catalytic sites of Escherichia coli ATP synthase

The role of αPhe-291 residue in phosphate binding by Escherichia coli F₁F₀-ATP synthase was examined. X-ray structures of bovine mitochondrial enzyme suggest that this residue resides in close proximity to the conserved βR246 residue. Herein, we show that mutations αF291D and αF291E in E. coli reduce the ATPase activity of F₁F₀ membranes by 350-fold. Yet, significant oxidative phosphorylation activity is retained. In contrast to wild-type, ATPase activities of mutants were not inhibited by MgADP-azide, MgADP-fluoroaluminate, or MgADP-fluoroscandium. Whereas, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) inhibited wild-type ATPase essentially completely, ATPase in mutants was inhibited maximally by ∼75%, although reaction still occurred at residue βTyr-297, proximal to αPhe-291 in the phosphate-binding pocket. Inhibition characteristics supported the conclusion that NBD-Cl reacts in βE (empty) catalytic sites, as shown previously by X-ray structure analysis. Phosphate protected against NBD-Cl inhibition in wild-type but not in mutants. In addition, our data suggest that the interaction of αPhe-291 with phosphate during ATP hydrolysis or synthesis may be distinct.     

Light-Dependent Incorporation of Adenine Nucleotide into Noncatalytic Sites of Chloroplast ATP Synthase

The binding of ADP and ATP to noncatalytic sites of dithiothreitol-modified chloroplast ATP synthase was studied. Selective binding of nucleotides to noncatalytic sites was provided by preliminary light incubation of thylakoid membranes with [¹⁴C]ADP followed by its dissociation from catalytic sites during dark ATP hydrolysis stimulated by bisulfite ions (“cold chase”). Incorporation of labeled nucleotides increased with increasing light intensity. Concentration-dependent equilibrium between free and bound nucleotides was achieved within 2–10 min with the following characteristic parameters: the maximal value of nucleotide incorporation was 1.5 nmol/mg of chlorophyll, and the dissociation constant was 1.5 μM. The dependence of nucleotide incorporation on Mg²⁺ concentration was slight and changed insignificantly upon substituting Ca²⁺ for Mg²⁺. Dissociation of nucleotide from noncatalytic sites was illumination dependent. The dissociation kinetics suggested the existence of at least two nucleotide-binding sites with different dissociation rate constants. __________ Translated from Biokhimiya, Vol. 70, No. 11, 2005, pp. 1514–1520. Original Russian Text Copyright ? 2005 by Malyan.     

Frontiers in ATP synthase research: Understanding the relationship between subunit movements and ATP Synthesis

How biological systems make ATP has intrigued many scientists for well over half the 20th century, and because of the importance and complexity of the problem it seems likely to continue to be a source of fascination to both senior and younger investigators well into the 21st century. Scientific battles fought to unravel the vast secrets by which ATP synthases work have been fierce, and great victories have been short-lived, tempered with the realization that more structures are needed, additional subunits remain to be conquered, and that during ATP synthesis, not one, but several subunits may undergo either significant conformational changes, repositioning, or perhaps even physical rotation similar to bacterial flagella^(1,2). In this introductory article, the author briefly summarizes our current knowledge about the complex substructure of ATP synthases, what we have learned from X-ray crystallography of the F₁ unit, and current evidence for subunit movements.     

gammaepsilon Sub-complex of thermophilic ATP synthase has the ability to bind ATP

The isolated epsilon subunit of F(1)-ATPase from thermophilic Bacillus PS3 (TF(1)) binds ATP [Y. Kato-Yamada, M. Yoshida, J. Biol. Chem. 278 (2003) 36013]. The obvious question is whether the ATP binding concern with the regulation of ATP synthase activity or not. If so, the epsilon subunit even in the ATP synthase complex should have the ability to bind ATP. To check if the ATP binding to the epsilon subunit within the ATP synthase complex may occur, the gammaepsilon sub-complex of TF(1) was prepared and ATP binding was examined. The results clearly showed that the gammaepsilon sub-complex can bind ATP.     

Improved purification for thermophilic F1F0 ATP synthase using n-dodecyl beta-D-maltoside

Here we report a fast, simple purification for thermophilic F1F0 ATP synthase (TF1F0) that utilizes a cocktail of stabilizing reagents and the detergent n-dodecyl beta-D-maltoside to yield enzyme with an ATPase activity of 41 micromol/min/mg, 2.5-fold higher than that previously reported. ATPase activity was 80% inhibited by the F0-reactive reagent dicyclohexylcarbodiimide, indicating that F1-F0 interactions were largely intact. To measure ATP-driven proton pumping activity, purified TF1F0 was incorporated into liposomes, and the ATP-induced change in internal pH was measured using the fluorescent probe pyranine. In the presence of valinomycin, a maximum ATP-driven deltapH of 0.8 units was obtained. To measure ATP synthesis activity, TF1F0 was incorporated into liposomes with the light-dependent proton pump bacteriorhodopsin. Proteoliposomes were illuminated to generate an electrochemical gradient, after which ADP and inorganic phosphate were added to initiate ATP synthesis. A steady state ATP synthesis activity of 490 nmol/min/mg was achieved after an initial approximately 30-min lag phase.     

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.     

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.     

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.     

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.     

Why Is the Mechanical Efficiency of F1-ATPase So High?

The experimentally measured mechanical efficiency of the F₁-ATPase under viscous loading is nearly 100%, far higher than any other hydrolysis-driven molecular motor (Yasuda et al., 1998). Here we give a molecular explanation for this remarkable property.     

Properties of Bound Inorganic Phosphate on Bovine Mitochondrial F1F0-ATP Synthase

Beef-heart mitochondrial F₁F₀-ATP synthase contained six molecules of bound inorganic phosphate (P_i). This phosphate exchanged completely with exogenous ³²P_i when the enzyme was exposed to 30% (v/v) dimethyl sulfoxide (DMSO) and then returned to a DMSO-free buffer (Beharry and Bragg 2001). Only two molecules were replaced by ³²P_i when the enzyme was not pretreated with DMSO. These two molecules of ³²P_i were not displaced from the enzyme by the treatment with 1 mM ATP. Similarly, two molecules of bound ³²P_i remained on the DMSO-pretreated enzyme following addition of ATP, that is, four molecules of ³²P_i were displaced by ATP. The ATP-resistant ³²P_i was removed from the enzyme by pyrophosphate. It is proposed that these molecules of ³²P_i are bound at an unfilled adenine nucleotide-binding noncatalytic site on the enzyme. Brief exposure of the enzyme loaded with two molecules of ³²P_i to DMSO, followed by removal of the DMSO, resulted in the loss of the bound ³²P_i and in the formation of two molecules of bound ATP from exogenous ADP. A third catalytic site on the enzyme was occupied by ATP, which could undergo a P_i ATP exchange reaction with bound P_i The presence of two catalytic sites containing bound P_i is consistent with the X-ray crystallographic structure of F₁ (Bianchet, et al., 1998). Thus, five of the six molecules of bound P_i were accounted for. Three molecules of bound P_i were at catalytic sites and participated in ATP synthesis or P_i ATP exchange. Two other molecules of bound P_i were present at a noncatalytic adenine nucleotide-binding site. The location and role of the remaining molecule of bound P_i remains to be established. We were unable to demonstrate, using chemical modification of sulfhydryl groups by iodoacetic acid, any gross difference in the conformation of F₁F₀ in DMSO-containing compared with DMSO-free buffers.     

ATP合酶的结构与催化机理

ATP合酶 (F1Fo 复合物) 是生物体内进行氧化磷酸化和光合磷酸化的关键酶.随着核磁共振、X射线晶体衍射、遗传学、化学交联等技术在ATP合酶研究中的广泛应用,ATP合酶的整体结构及其各组成亚基结构的研究都有很大的进展.其中细菌ATP合酶结构的研究更为深入.目前对质子通过Fo的转运方式提出两种模型:单通道和双半通道模型.对扭力矩的形成以及旋转催化也有了进一步的认识.Boyer提出的结合改变机理推动了ATP合酶催化机制的研究,现在主要有两点催化机制和三点催化机制.ATP合酶的催化反应受酶的构象变化和外在条件的调节.     

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.     

ATP synthases—Structure of the F1-moiety and its relationship to function and mechanism

A great deal of progress has been made in understanding both the structure and the mechanism of F₁-ATPase. The primary structure is now fully known for at least five species. Sequence comparison between chloroplast, photobacteria, aerobic bacteria, and mitochondrial representatives allow us to infer more general functional relationships and evolutionary trends. Although the F₁ moiety is the most studied segment of the H⁺-ATPase complex, there is not a full understanding of the mechanism and regulation of its hydrolytic activity. The subunit is now known to contain one and probably two nucleotide binding domains, one of which is believed to be a catalytic site. Recently, two similar models have been proposed to attempt to describe the active part of the subunits. These models are mainly an attempt to use the structure of adenylate kinase to represent a more general working model for nucleotide binding phosphotransferases. Labelling experiments seem to indicate that several critical residues outside the region described by the adenylate kinase part of this model are also actively involved in the ATPase activity. New models will have to be introduced to include these regions. Finally, it seems that a consensus has been reached with regard to a broad acceptance of the asymmetric structure of the F₁-moiety. In addition, recent experimental evidence points toward the presence of nonequivalent subunits to describe the functional activity of the F₁-ATPase. A summary diagram of the conformational and binding states of the enzyme including the nonequivalent subunit is presented. Additional research is essential to establish the role of the minor subunits—and of the asymmetry they introduce in F₁—on the physiological function of the enzyme.     

Inhibitory and Anchoring Domains in the ATPase Inhibitor Protein IF1 of Bovine Heart Mitochondrial ATP Synthase

The inhibitor protein IF1 is a basic protein of 84 residues which inhibits the ATPase activity of the mitochondrial FoF1-ATP synthase complex without having any effect on ATP synthesis. Results of cross-linking and limited proteolysis experiments are presented showing that in the intact FoF1 complex "in situ," in the inner membrane of bovine heart mitochondria, the central segment of IF1 (residues 42-58) binds to the alpha and beta subunits of F1 in a pH dependent process, and inhibits the ATPase activity. The C-terminal region of IF1 binds, simultaneously, to the OSCP subunit of Fo in a pH-independent process. This binding keeps IF1 anchored to the complex, both under inhibitory conditions, at acidic pH, and noninhibitory conditions at alkaline pH.