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.     

The Catalytic Transition State in ATP Synthase

The catalytic transition state of ATP synthase has been characterized and modeled by combined use of (1) Mg-ADP–fluoroaluminate, Mg-ADP–fluoroscandium, and corresponding Mg-IDP–fluorometals as transition-state analogs; (2) fluorescence signals of -Trp331 and -Trp148 as optical probes to assess formation of the transition state; (3) mutations of critical catalytic residues to determine side-chain ligands required to stabilize the transition state. Rate acceleration by positive catalytic site cooperativity is explained as due to mobility of -Arg376, acting as an arginine finger residue, which interacts with nucleotide specifically at the transition state step of catalysis, not with Mg-ATP- or Mg-ADP-bound ground states. We speculate that formation and collapse of the transition state may engender catalytic site / subunit-interface conformational movement, which is linked to -subunit rotation.     

The Phylogenetic Signature Underlying ATP Synthase c-Ring Compliance

The proton-driven ATP synthase (F_OF₁) is comprised of two rotary, stepping motors (F_O and F₁) coupled by an elastic power transmission. The elastic compliance resides in the rotor module that includes the membrane-embedded F_O c-ring. Proton transport by F_O is firmly coupled to the rotation of the c-ring relative to other F_O subunits (ab₂). It drives ATP synthesis. We used a computational method to investigate the contribution of the c-ring to the total elastic compliance. We performed principal component analysis of conformational ensembles built using distance constraints from the bovine mitochondrial c-ring x-ray structure. Angular rotary twist, the dominant ring motion, was estimated to show that the c-ring accounted in part for the measured compliance. Ring rotation was entrained to rotation of the external helix within each hairpin-shaped c-subunit in the ring. Ensembles of monomer and dimers extracted from complete c-rings showed that the coupling between collective ring and the individual subunit motions was independent of the size of the c-ring, which varies between organisms. Molecular determinants were identified by covariance analysis of residue coevolution and structural-alphabet-based local dynamics correlations. The residue coevolution gave a readout of subunit architecture. The dynamic couplings revealed that the hinge for both ring and subunit helix rotations was constructed from the proton-binding site and the adjacent glycine motif (IB-GGGG) in the midmembrane plane. IB-GGGG motifs were linked by long-range couplings across the ring, while intrasubunit couplings connected the motif to the conserved cytoplasmic loop and adjacent segments. The correlation with principal collective motions shows that the couplings underlie both ring rotary and bending motions. Noncontact couplings between IB-GGGG motifs matched the coevolution signal as well as contact couplings. The residue coevolution reflects the physiological importance of the dynamics that may link proton transfer to ring compliance.     

The b Subunit of Escherichia coli ATP Synthase

The b subunit of ATP synthase is a major component of the second stalk connecting the F₁and F₀ sectors of the enzyme and is essential for normal assembly and function. The156-residue b subunit of the Escherichia coli ATP synthase has been investigated extensivelythrough mutagenesis, deletion analysis, and biophysical characterization. The two copies ofb exist as a highly extended, helical dimer extending from the membrane to near the top ofF₁, where they interact with the subunit. The sequence has been divided into four domains:the N-terminal membrane-spanning domain, the tether domain, the dimerization domain, andthe C-terminal -binding domain. The dimerization domain, contained within residues 60–122,has many properties of a coiled-coil, while the -binding domain is more globular. Sites ofcrosslinking between b and the a, , , and subunits of ATP synthase have been identified,and the functional significance of these interactions is under investigation. The b dimer mayserve as an elastic element during rotational catalysis in the enzyme, but also directly influencesthe catalytic sites, suggesting a more active role in coupling.     

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.     

The Oligomycin Axis of Mitochondrial ATP Synthase: OSCP and the Proton Channel

Oligomycin has long been known as an inhibitor of mitochondrial ATP synthase, putatively binding the F_o subunits 9 and 6 that contribute to proton channel function of the complex. As its name implies, OSCP is the oligomycin sensitivity-conferring protein necessary for the intact enzyme complex to display sensitivity to oligomycin. Recent advances concerning the structure and mechanism of mitochondrial ATP synthase have led to OSCP now being considered a component of the peripheral stator stalk rather than a central stalk component. How OSCP confers oligomycin sensitivity on the enzyme is unknown, but probably reflects important protein–protein interactions made within the assembled complex and transmitted down the stator stalk, thereby influencing proton channel function. We review here our studies directed toward establishing the stoichiometry, assembly, and function of OSCP in the context of knowledge of the organization of the stator stalk and the proton channel.     

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.     

Cryo-EM Structure of the Yeast ATP Synthase

We have used electron cryomicroscopy of single particles to determine the structure of the ATP synthase from Saccharomyces cerevisiae. The resulting map at 24 Å resolution can accommodate atomic models of the F₁-c₁₀ subcomplex, the peripheral stalk subcomplex, and the N-terminal domain of the oligomycin sensitivity conferral protein. The map is similar to an earlier electron cryomicroscopy structure of bovine mitochondrial ATP synthase but with important differences. It resolves the internal structure of the membrane region of the complex, especially the membrane embedded subunits b, c, and a. Comparison of the yeast ATP synthase map, which lacks density from the dimer-specific subunits e and g, with a map of the bovine enzyme that included e and g indicates where these subunits are located in the intact complex. This new map has allowed construction of a model of subunit arrangement in the F_O motor of ATP synthase that dictates how dimerization of the complex via subunits e and g might occur.     

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.     

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.     

The efficiency of ATP synthase as a molecular machine

On the basis of the experimentally measured isotope effect of magnesium (25)Mg on the rate of ATP synthesis by mithochondrial ATP synthase, the rate constants of the reactions in the catalytic site have been estimated. The limiting step of the synthesis is shown to be the addition of8phosphoryl anion-radical of ADP to the P=O bond of phosphate with the rate constant of 1,2 x 10(8) s(-1). From the ratio of the rate constants, the mechanochemical efficiency of ATP synthase was estimated to be about 10% for enzymes with (24, 26)Mg isotopes and to be doubled for enzymes with (25)Mg in the catalytic site.     

The ATP Synthase atpHAGDC (F1) Operon from Rhodobacter capsulatus

The atpHAGDC operon of Rhodobacter capsulatus, containing the five genes coding for the F₁ sector of the ATP synthase, has been cloned and sequenced. The promoter region has been defined by primer extension analysis. It was not possible to obtain viable cells carrying atp deletions in the R. capsulatus chromosome, indicating that genes coding for ATP synthase are essential, at least under the growth conditions tested. We were able to circumvent this problem by combining gene transfer agent transduction with conjugation. This method represents an easy way to construct strains carrying mutations in indispensable genes.     

Genes of Human ATP Synthase: Their Roles in Physiology and Aging

The reaction of ATP synthase (F₀F₁) is the final step in oxidative phosphorylation (OXPHOS). Although OXPHOS has been studied extensively in bacteria, no tissue-specific functions nor bioenergetic disease, such as mitochondrial encephalomyopathy and aging occur in these organisms. Recent developments of the Human Genome Project will become an important factor in the study of mammalian bioenergetics. To elucidate the physiological roles of human F₀F₁, genes encoding the subunits of F₀F₁ were sequenced, and their expression in human cells was analyzed. The following results were obtained: A. The roles of the residues in F₀F₁ are not only to transform the energy of the electrochemical potential (H⁺) across the membrane, but also to respond rapidly to the changes in the energy demand by regulating the intramolecular rotation of F₀F₁ with the H⁺ and the inhibitors of the ATPase. B. The roles of the control regions of the F₀F₁ genes, are to coordinate both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) depending on the energy demand of the cells, especially in muscle. C. The cause of the age-dependent decline of ATP synthesis has been attributed to the accumulation of mutations in mtDNA. However, the involvement of nDNA in the decline is also important because of telomere shortening in somatic cells, and age-dependent mtDNA expression analyzed with ° cells (cells without mtDNA).     

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.     

Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth

We examined the thymoquinone induced inhibition of purified F₁ or membrane bound F₁F_O E. coli ATP synthase. Both purified F₁ and membrane bound F₁F_O were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F₁F_o and purified F₁ preparations. Moreover, thymoquinone induced inhibition of ATP synthase expressing wild-type E. coli cell growth and non-inhibition of ATPase gene deleted null control cells demonstrates that ATP synthase is a molecular target for thymoquinone. This also links the beneficial dietary based antimicrobial and anticancer effects of thymoquinone to its inhibitory action on ATP synthase.     

Toward an Adequate Scheme for the ATP Synthase Catalysis

The suggestions from the author's group over the past 25 years for how steps in catalysis by ATP synthase occur are reviewed. Whether rapid ATP hydrolysis requires the binding of ATP to a second site (bi-site activation) or to a second and third site (tri-site activation) is considered. Present evidence is regarded as strongly favoring bi-site activation. Presence of nucleotides at three sites during rapid ATP hydrolysis can be largely accounted for by the retention of ADP formed and/or by the rebinding of ADP formed. Menz, Leslie and Walker ((2001) FEBS Lett., 494, 11-14) recently attained an X-ray structure of a partially closed enzyme form that binds ADP better than ATP. This accomplishment and other considerations form the base for a revised reaction sequence. Three types of catalytic sites are suggested, similar to those proposed before the X-ray data became available. During net ATP synthesis a partially closed site readily binds ADP and P_i but not ATP. At a closed site, tightly bound ADP and P_i are reversibly converted to tightly bound ATP. ATP is released from a partially closed site that can readily bind ATP or ADP. ATP hydrolysis when protonmotive force is low or lacking occurs simply by reversal of all steps with the opposite rotation of the subunit. Each type of site can exist in various conformations or forms as they are interconverted during a 120° rotation. The conformational changes with the ATP synthase, including the vital change when bound ADP and P_i are converted to bound ATP, are correlated with those that occur in enzyme catalysis in general, as illustrated by recent studies of Rose with fumarase. The _B structure of Walker's group is regarded as an unlikely, or only quite transient, intermediate. Other X-ray structures are regarded as closely resembling but not identical with certain forms participating in catalysis. Correlation of the suggested reaction scheme with other present information is considered.     

Lineage-Specific Evolution of Echinoderm Mitochondrial ATP Synthase Subunit 8

Peculiar evolutionary properties of the subunit 8 of mitochondrial ATP synthase (ATPase8) are revealed by comparative analyses carried out between both closely and distantly related species of echinoderms. The analysis of nucleotide substitution in the three echinoids demonstrated a relaxation of amino acid functional constraints. The deduced protein sequences display a well conserved domain at the N-terminus, while the central part is very variable. At the C-terminus, the broad distribution of positively charged amino acids, which is typical of other organisms, is not conserved in the two different echinoderm classes of the sea urchins and of the sea stars. Instead, a motif of three amino acids, so far not described elsewhere, is conserved in sea urchins and is found to be very similar to the motif present in the sea stars. Our results indicate that the N-terminal region seems to follow the same evolutionary pattern in different organisms, while the maintenance of the C-terminal part in a phylum-specific manner may reflect the co-evolution of mitochondrial and nuclear genes.     

Subunit f of the Yeast Mitochondrial ATP Synthase: Topological and Functional Studies

Modified versions of subunit f were produced by mutagenesis of theATP17 gene of Saccharomyces cerevisiae. A version of subunit f devoid of thelast 28 amino acid residues including the unique transmembranous domaincomplemented the oxidative phosphorylation of the null mutant. However, atwo-fold decrease in the specific ATP synthase activity was measured andattributed to a decrease in the stability of the mutant ATP synthase complexas shown by the low oligomycin-sensitive ATPase activity at alkaline pH. Themodification or not by non-permeant maleimide reagents of cysteine residuesintroduced at the N and C termini of subunit f indicated aN_in-C_out orientation. From the C terminus of subunit fit was possible to cross-link subunit 4 (also called subunit b), which isanother component of the F₀ sector and which also displays a shorthydrophilic segment exposed to the intermembrane space.