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1.
The ATP synthase of many archaea has the conserved sodium ion binding motif in its rotor subunit, implying that these A1AO-ATP synthases use Na+ as coupling ion. However, this has never been experimentally verified with a purified system. To experimentally address the nature of the coupling ion, we have purified the A1AO-ATP synthase from T. onnurineus. It contains nine subunits that are functionally coupled. The enzyme hydrolyzed ATP, CTP, GTP, UTP, and ITP with nearly identical activities of around 40 units/mg of protein and was active over a wide pH range with maximal activity at pH 7. Noteworthy was the temperature profile. ATP hydrolysis was maximal at 80 °C and still retained an activity of 2.5 units/mg of protein at 45 °C. The high activity of the enzyme at 45 °C opened, for the first time, a way to directly measure ion transport in an A1AO-ATP synthase. Therefore, the enzyme was reconstituted into liposomes generated from Escherichia coli lipids. These proteoliposomes were still active at 45 °C and coupled ATP hydrolysis to primary and electrogenic Na+ transport. This is the first proof of Na+ transport by an A1AO-ATP synthase and these findings are discussed in light of the distribution of the sodium ion binding motif in archaea and the role of Na+ in the bioenergetics of archaea.  相似文献   

2.
3.
In eukaryotic and prokaryotic cells, F-ATP synthases provide energy through the synthesis of ATP. The chloroplast F-ATP synthase (CF1FO-ATP synthase) of plants is integrated into the thylakoid membrane via its FO-domain subunits a, b, b’ and c. Subunit c with a stoichiometry of 14 and subunit a form the gate for H+-pumping, enabling the coupling of electrochemical energy with ATP synthesis in the F1 sector.Here we report the crystallization and structure determination of the c14-ring of subunit c of the CF1FO-ATP synthase from spinach chloroplasts. The crystals belonged to space group C2, with unit-cell parameters a=144.420, b=99.295, c=123.51 Å, and β=104.34° and diffracted to 4.5 Å resolution. Each c-ring contains 14 monomers in the asymmetric unit. The length of the c-ring is 60.32 Å, with an outer ring diameter 52.30 Å and an inner ring width of 40 Å.  相似文献   

4.
《BBA》1987,893(2):275-288
The membrane-bound ATP synthase from chloroplasts can occur in different redox and activation states. In the absence of reductants the enzyme usually is oxidized and inactive, Eoxi. Illumination in the presence of dithiothreitol leads to an active, reduced enzyme, Ereda. If this form is stored in the dark in the presence of dithiothreitol an inactive, reduced enzyme Eredi is formed. The rates of ATP synthesis and ATP hydrolysis catalyzed by the different enzyme species are measured as a function of ΔpH (Δψ = 0 mV). The ΔpH was generated with an acid-base transition using a rapid-mixing quenched flow apparatus. The following results were obtained. (1) The oxidized ATP synthase catalyzes high rates of ATP synthesis, voxmax = 400 ATP per CF0F1 per s. The half-maximal rate is obtained at ΔpH = 3.4. (2) The active, reduced ATP synthase catalyzes high rates of ATP synthesis, vredmax = 400 ATP per CF0F1 per s. The half-maximal rate is obtained at ΔpH = 2.7. It catalyzes also high rates of ATP hydrolysis vredmax = −90 ATP per CF0F per s at ΔpH = 0. (3) The inactive species (both oxidized and reduced) catalyze neither ATP synthesis nor ATP hydrolysis. The activation/inactivation of the reduced enzyme is completely reversible. (4) The activation of the reduced, inactive enzyme is measured as a function of ΔpH by measuring the rate of ATP hydrolysis catalyzed by the active species. Half-maximal activation is observed at ΔpH = 2.2. (5) On the basis of these results a reaction scheme is proposed relating the redox reaction, the activation and the catalytic reaction of the chloroplast ATP synthase.  相似文献   

5.
Extracellular ATP formation from ADP and inorganic phosphate, attributed to the activity of a cell surface ATP synthase, has so far only been reported in cultures of some proliferating and tumoral cell lines. We now provide evidence showing the presence of a functionally active ecto-FoF1-ATP synthase on the plasma membrane of normal tissue cells, i.e. isolated rat hepatocytes. Both confocal microscopy and flow cytometry analysis show the presence of subunits of F1 (α/β and γ) and Fo (FoI-PVP(b) and OSCP) moieties of ATP synthase at the surface of rat hepatocytes. This finding is confirmed by immunoblotting analysis of the hepatocyte plasma membrane fraction. The presence of the inhibitor protein IF1 is also detected on the hepatocyte surface. Activity assays show that the ectopic-ATP synthase can work both in the direction of ATP synthesis and hydrolysis. A proton translocation assay shows that both these mechanisms are accompanied by a transient flux of H+ and are inhibited by F1 and Fo-targeting inhibitors. We hypothesise that ecto-FoF1-ATP synthase may control the extracellular ADP/ATP ratio, thus contributing to intracellular pH homeostasis.  相似文献   

6.
Alexander Wiedenmann 《BBA》2008,1777(10):1301-1310
The membrane-embedded F0 part of ATP synthases is responsible for ion translocation during ATP synthesis and hydrolysis. Here, we describe an in vitro system for measuring proton fluxes through F0 complexes by fluorescence changes of the entrapped fluorophore pyranine. Starting from purified enzyme, the F0 part was incorporated unidirectionally into phospholipid vesicles. This allowed analysis of proton transport in either synthesis or hydrolysis direction with Δψ or ΔpH as driving forces. The system displayed a high signal-to-noise ratio and can be accurately quantified. In contrast to ATP synthesis in the Escherichia coli F1F0 holoenzyme, no significant difference was observed in the efficiency of ΔpH or Δψ as driving forces for H+-transport through F0. Transport rates showed linear dependency on the driving force. Proton transport in hydrolysis direction was about 2400 H+/(s × F0) at Δψ of 120 mV, which is approximately twice as fast as in synthesis direction. The chloroplast enzyme was faster and catalyzed H+-transport at initial rates of 6300 H+/(s × F0) under similar conditions. The new method is an ideal tool for detailed kinetic investigations of the ion transport mechanism of ATP synthases from various organisms.  相似文献   

7.
The anaerobic bacterium Fusobacterium nucleatum uses glutamate decarboxylation to generate a transmembrane gradient of Na+. Here, we demonstrate that this ion-motive force is directly coupled to ATP synthesis, via an F1Fo-ATP synthase with a novel Na+ recognition motif, shared by other human pathogens. Molecular modeling and free-energy simulations of the rotary element of the enzyme, the c-ring, indicate Na+ specificity in physiological settings. Consistently, activity measurements showed Na+ stimulation of the enzyme, either membrane-embedded or isolated, and ATP synthesis was sensitive to the Na+ ionophore monensin. Furthermore, Na+ has a protective effect against inhibitors targeting the ion-binding sites, both in the complete ATP synthase and the isolated c-ring. Definitive evidence of Na+ coupling is provided by two identical crystal structures of the c11 ring, solved by X-ray crystallography at 2.2 and 2.6 Å resolution, at pH 5.3 and 8.7, respectively. Na+ ions occupy all binding sites, each coordinated by four amino acids and a water molecule. Intriguingly, two carboxylates instead of one mediate ion binding. Simulations and experiments demonstrate that this motif implies that a proton is concurrently bound to all sites, although Na+ alone drives the rotary mechanism. The structure thus reveals a new mode of ion coupling in ATP synthases and provides a basis for drug-design efforts against this opportunistic pathogen.  相似文献   

8.
The molecular mechanism by which the membrane-embedded FO sector of the mitochondrial ATP synthase translocates protons, thus dissipating the transmembrane protonmotive force and leading to ATP synthesis, involves the neutralization of the carboxylate residues of the c-ring. Carboxylates are thought to constitute the binding sites for ion translocation. In order to cast light on this mechanism, we exploited N,N’-dicyclohexylcarbodiimide, which covalently binds to FO c-ring carboxylates, and ionophores which selectively modulate the transmembrane electric (Δφ) and chemical (ΔpH) gradients such as valinomycin, nigericin and dinitrophenol. ATP hydrolysis was evaluated in mitochondrial preparations and/or inside-out submitochondrial particles from mussel and mammalian tissues under different experimental conditions. The experiments pointed out striking similarities between mussel and mammalian mitochondrial ATP synthase. Our results support the hypothesis that the ATP synthase of Mytilus galloprovincialis induces intersubunit torque generation and translocates H+ by coordinating the hydronium ion (H3O+) in the ion binding site of FO. Our results are consistent with the hypothesis that in mussel mitochondria the main component of the electrochemical gradient driving proton flux and ATP synthesis is Δφ. Therefore, mussel FO probably contains a small c-ring, which implies a low bioenergetic cost of making ATP as in mammals. These features which make mussel mitochondria as efficient in ATP production as mammalian ones may be especially advantageous in facultative aerobic species which intermittently exploit mitochondrial respiration to generate ATP.  相似文献   

9.
Silke Schmidt 《BBA》2009,1787(6):691-952
The acetogenic bacterium Acetobacterium woodii uses a transmembrane electrochemical sodium ion potential for bioenergetic reactions. A primary sodium ion potential is established during carbonate (acetogenesis) as well as caffeate respiration. The electrogenic Na+ pump connected to the Wood-Ljungdahl pathway (acetogenesis) still remains to be identified. The pathway of caffeate reduction with hydrogen as electron donor was investigated and the only membrane-bound activity was found to be a ferredoxin-dependent NAD+ reduction. This exergonic electron transfer reaction may be catalyzed by the membrane-bound Rnf complex that was discovered recently and is suggested to couple exergonic electron transfer from ferredoxin to NAD+ to the vectorial transport of Na+ across the cytoplasmic membrane. Rnf may also be involved in acetogenesis. The electrochemical sodium ion potential thus generated is used to drive endergonic reactions such as flagellar rotation and ATP synthesis. The ATP synthase is a member of the F1FO class of enzymes but has an unusual and exceptional feature. Its membrane-embedded rotor is a hybrid made of FO and VO-like subunits in a stoichiometry of 9:1. This stoichiometry is apparently not variable with the growth conditions. The structure and function of the Rnf complex and the Na+ F1FO ATP synthase as key elements of the Na+ cycle in A. woodii are discussed.  相似文献   

10.
A correlation between the rate of ATP synthesis by F0F1 ATP synthase and formate oxidation by formate hydrogen lyase (FHL) has been found in inside-out membrane vesicles of the Escherichia coli mutant JW 136 (Δhyahyb) with double deletions of hydrogenases 1 and 2, grown anaerobically on glucose in the absence of external electron acceptors at pH 6.5. ATP synthesis was suppressed by the H+-ATPase inhibitors N,N′-dicyclohexylcarbodiimide, sodium azide, and the uncoupler carbonyl cyanide m-chlorophenylhydrazone. Copper ions inhibited formate-dependent hydrogenase and ATP-synthase activities but did not affect the ATPase activity of the vesicles. The maximal rate of ATP synthesis (0.83 μmol/min per mg protein) was determined at simultaneous application of sodium formate, ADP, and inorganic phosphate, and was stimulated by K+ ions. The results confirm the assumption of a dual role of hydrogenase 3, the formate hydrogen lyase subunit that can couple the reduction of protons to H2 and their translocation through membrane with chemiosmotic synthesis of ATP.  相似文献   

11.
H+-FOF1-ATP synthase couples proton flow through its membrane portion, FO, 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.  相似文献   

12.
For functional characterization, we isolated the F1FO-ATP synthase of the thermophilic cyanobacterium Thermosynechococcus elongatus. Because of the high content of phycobilisomes, a combination of dye-ligand chromatography and anion exchange chromatography was necessary to yield highly pure ATP synthase. All nine single F1FO subunits were identified by mass spectrometry. Western blotting revealed the SDS stable oligomer of subunits c in T. elongatus. In contrast to the mass archived in the database (10,141 Da), MALDI-TOF-MS revealed a mass of the subunit c monomer of only 8238 Da. A notable feature of the ATP synthase was its ability to synthesize ATP in a wide temperature range and its stability against chaotropic reagents. After reconstitution of F1FO into liposomes, ATP synthesis energized by an applied electrochemical proton gradient demonstrated functional integrity. The highest ATP synthesis rate was determined at the natural growth temperature of 55 °C, but even at 95 °C ATP production occurred. In contrast to other prokaryotic and eukaryotic ATP synthases which can be disassembled with Coomassie dye into the membrane integral and the hydrophilic part, the F1FO-ATP synthase possessed a particular stability. Also with the chaotropic reagents sodium bromide and guanidine thiocyanate, significantly harsher conditions were required for disassembly of the thermophilic ATP synthase.  相似文献   

13.
《BBA》2006,1757(5-6):304-310
Fo·F1-ATP synthase in inside-out coupled vesicles derived from Paracoccus denitrificans catalyzes Pi-dependent proton-translocating ATPase reaction if exposed to prior energization that relieves ADP·Mg2+-induced inhibition (Zharova, T.V. and Vinogradov, A.D. (2004) J. Biol. Chem.,279, 12319–12324). Here we present evidence that the presence of medium ADP is required for the steady-state energetically self-sustained coupled ATP hydrolysis. The initial rapid ATPase activity is declined to a certain level if the reaction proceeds in the presence of the ADP-consuming, ATP-regenerating system (pyruvate kinase/phosphoenol pyruvate). The rate and extent of the enzyme de-activation are inversely proportional to the steady-state ADP concentration, which is altered by various amounts of pyruvate kinase at constant ATPase level. The half-maximal rate of stationary ATP hydrolysis is reached at an ADP concentration of 8 × 10−6 M. The kinetic scheme is proposed explaining the requirement of the reaction products (ADP and Pi), the substrates of ATP synthesis, in the medium for proton-translocating ATP hydrolysis by P. denitrificans Fo·F1-ATP synthase.  相似文献   

14.
The Na+ F1FO ATP synthase of the anaerobic, acetogenic bacterium Acetobacterium woodii has a unique FOVO hybrid rotor that contains nine copies of a FO-like c subunit and one copy of a VO-like c 1 subunit with one ion binding site in four transmembrane helices whose cellular function is obscure. Since a genetic system to address the role of different c subunits is not available for this bacterium, we aimed at a heterologous expression system. Therefore, we cloned and expressed its Na+ F1FO ATP synthase operon in Escherichia coli. A Δatp mutant of E. coli produced a functional, membrane-bound Na+ F1FO ATP synthase that was purified in a single step after inserting a His6-tag to its β subunit. The purified enzyme was competent in Na+ transport and contained the FOVO hybrid rotor in the same stoichiometry as in A. woodii. Deletion of the atpI gene from the A. woodii operon resulted in a loss of the c ring and a mis-assembled Na+ F1FO ATP synthase. AtpI from E. coli could not substitute AtpI from A. woodii. These data demonstrate for the first time a functional production of a FOVO hybrid rotor in E. coli and revealed that the native AtpI is required for assembly of the hybrid rotor.  相似文献   

15.
We prepared ATP photosynthetic vesicles from inside-out membranes of Escherichia coli cells that express delta-rhodopsin (a novel light-driven H+ transporter) and TF0F1-ATP synthase (a thermo-stable ATP synthase). These vesicles showed light-dependent ATP synthesis. Furthermore, coupling the ATP photosynthetic vesicles with an ATP-hydrolyzing hexokinase enabled light-dependent glucose consumption. The ATP photosynthetic vesicles indicate their potential to applied to light-driven ATP-regenerating bioprocess for various ATP-hydrolyzing bioproductions.  相似文献   

16.
Incubation of the isolated H+-ATPase from chloroplasts, CF0F1, with 2-azido-[α-32P]ATP leads to the binding of this nucleotide to different sites. These sites were identified after removal of free nucleotides, UV-irradiation and trypsin treatment by separation of the tryptic peptides by ion exchange chromatography. The nitreno-AMP, nitreno-ADP and nitreno-ATP peptides were further separated on a reversed phase column, the main fractions were subjected to amino acid sequence analysis and the derivatized tyrosines were used to distinguish between catalytic (β-Tyr362) and non-catalytic (β-Tyr385) sites. Several incubation procedures were developed which allow a selective occupation of each of the three non-catalytic sites. The non-catalytic site with the highest dissociation constant (site 6) becomes half maximally filled at 50 μM 2-azido-[α-32P]ATP, that with the intermediate dissociation constant (site 5) at 2 μM. The ATP at the site with the lowest dissociation constant had to be hydrolyzed first to ADP before a replacement by 2-azido-[α-32P]ATP was possible. CF0F1 with non-covalently bound 2-azido-[α-32P]ATP and after covalent derivatization was reconstituted into liposomes and the rates of ATP synthesis as well as ATP hydrolysis were measured after energization of the proteoliposomes by ΔpH/Δϕ. Non-covalent binding of 2-azido-ATP to any of the three non-catalytic sites does not influence ATP synthesis and ATP hydrolysis, whereas covalent derivatization of any of the three sites inhibits both, the degree being proportional to the degree of derivatization. Extrapolation to complete inhibition indicates that derivatization of one site (either 4 or 5 or 6) is sufficient to block completely multi-site catalysis. The rates of ATP synthesis and ATP hydrolysis were measured as a function of the ADP and ATP concentration from uni-site to multi-site conditions with covalently derivatized and non-derivatized CF0F1. Uni-site ATP synthesis and ATP hydrolysis were not inhibited by covalent derivatization of any of the non-catalytic sites, whereas multi-site catalysis is inhibited. These results indicate that multi-site catalysis requires some flexibility between β- and α-subunits which is abolished by covalent derivatization of β-Tyr385 with a 2-nitreno-adenine nucleotide. Conformational changes connected with energy transduction between the F0-part and the F1-part are either not required for uni-site ATP synthesis or they are not impaired by the derivatization of any of the three β-Tyr385.  相似文献   

17.
Vertebrate retinal rod outer segments (OS) consist of a stack of disks surrounded by the plasma membrane, where phototransduction takes place. Energetic metabolism in rod OS remains obscure. Literature described a so-called Mg2+-dependent ATPase activity, while our previous results demonstrated the presence of oxidative phosphorylation (OXPHOS) in OS, sustained by an ATP synthetic activity. Here we propose that the OS ATPase and ATP synthase are the expression of the same protein, i.e., of F1Fo-ATP synthase. Imaging on bovine retinal sections showed that some OXPHOS proteins are expressed in the OS. Biochemical data on bovine purified rod OS, characterized for purity, show an ATP synthase activity, inhibited by classical F1Fo-ATP synthase inhibitors. Moreover, OS possess a pH-dependent ATP hydrolysis, inhibited by pH values below 7, suggestive of the functioning of the inhibitor of F1 (IF1) protein. WB confirmed the presence of IF1 in OS, substantiating the expression of F1Fo ATP synthase in OS. Data suggest that the OS F1Fo ATP synthase is able to hydrolyze or synthesize ATP, depending on in vitro or in vivo conditions and that the role of IF1 would be pivotal in the prevention of the reversal of ATP synthase in OS, for example during hypoxia, granting photoreceptor survival.  相似文献   

18.
The ϵ subunit of bacterial FoF1-ATP synthase (FoF1), a rotary motor protein, is known to inhibit the ATP hydrolysis reaction of this enzyme. The inhibitory effect is modulated by the conformation of the C-terminal α-helices of ϵ, and the “extended” but not “hairpin-folded” state is responsible for inhibition. Although the inhibition of ATP hydrolysis by the C-terminal domain of ϵ has been extensively studied, the effect on ATP synthesis is not fully understood. In this study, we generated an Escherichia coli FoF1 (EFoF1) mutant in which the ϵ subunit lacked the C-terminal domain (FoF1ϵΔC), and ATP synthesis driven by acid-base transition (ΔpH) and the K+-valinomycin diffusion potential (ΔΨ) was compared in detail with that of the wild-type enzyme (FoF1ϵWT). The turnover numbers (kcat) of FoF1ϵWT were severalfold lower than those of FoF1ϵΔC. FoF1ϵWT showed higher Michaelis constants (Km). The dependence of the activities of FoF1ϵWT and FoF1ϵΔC on various combinations of ΔpH and ΔΨ was similar, suggesting that the rate-limiting step in ATP synthesis was unaltered by the C-terminal domain of ϵ. Solubilized FoF1ϵWT also showed lower kcat and higher Km values for ATP hydrolysis than the corresponding values of FoF1ϵΔC. These results suggest that the C-terminal domain of the ϵ subunit of EFoF1 slows multiple elementary steps in both the ATP synthesis/hydrolysis reactions by restricting the rotation of the γ subunit.  相似文献   

19.
Ronald S. Kaplan  P.S. Coleman 《BBA》1978,501(2):269-274
1. The use of 1,N6-ethenoadenosine 5′-triphosphate (?-ATP), a synthetic, fluorescent analog of ATP, by whole rat liver mitochondria and by submitochondrial particles produced via sonication has been studied.2. Direct [3H]adenine nucleotide uptake studies with isolated mitochondria, indicate the ?-[3H]ATP is not transported through the inner membrane by the adenine nucleotide carrier and is therefore not utilized by the 2,4-dinitrophenol-sensitive F1-ATPase (EC 3.6.1.3) that functions in oxidative phosphorylation. However, ?-ATP is hydrolyzed by a Mg2+-dependent, 2,4-dinitrophenol-insensitive ATPase that is characteristic of damaged mitochondria.3. ?-ATP can be utilized quite well by the exposed F1-ATPase of sonic submitochondrial particles. This ?-ATP hydrolysis activity is inhibited by oligomycin and stimulated by 2,4-dinitrophenol. The particle F1-ATPase displays similar Km values for both ATP and ?-ATP; however, the V with ATP is approximately six times greater than with ?-ATP.4. Since ?-ATP is a capable substrate for the submitochondrial particle F1-ATPase, it is proposed that the fluorescent properties of this ATP analog might be employed to study the submitochondrial particle F1-ATPase complex, and its response to various modifiers of oxidative phosphorylation.  相似文献   

20.
Kinetic models of the F0F1-ATPase able to transport H+ or/and Na+ ions are proposed. It is assumed that (i) H+ and Na+ compete for the same binding sites, (ii) ion translocation through F0 is coupled to the rate-limiting step of the F1-catalyzed reaction. The main characteristics of the dependences of ATP synthesis and hydrolysis rates on Δφ, ΔpH, and ΔpNa are predicted for various versions of the coupling model. The mechanism of the switchover from \(\Delta \bar \mu _{H^ + } \) -dependent synthesis to the \(\Delta \bar \mu _{Na^ + } \) -dependent one is demonstrated. It is shown that even with a drastic drop in \(\Delta \bar \mu _{H^ + } \) , ATP hydrolysis by the proton mode of catalysis can be effectively inhibited by Δφ and ΔpNa. The results obtained strongly support the possibility that the same F0F1-ATPase in bacterial cells can utilize both \(\Delta \bar \mu _{H^ + } \) and \(\Delta \bar \mu _{Na^ + } \) for ATP synthesis underin vivo conditions.  相似文献   

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