Paola Turina

TOPOGRAPHICAL STRUCTURE OF MEMBRANE-BOUND ESCHERICHIA COLI F1F0 ATP SYNTHASE IN AQUEOUS BUFFER

Scanning force microscope images of membrane‐bound Escherichia coli ATP synthase F0 complexes have been obtained in aqueous solution. The images show a consistent set of internal features: a ring structure which surrounds a central dimple and contains an asymmetric lateral mass. Images of trypsin‐treated F0 complexes, which have lost part of their b subunits, show a reduced asymmetric mass, while images of c‐subunit oligomers, which lack both the a and b subunits, show a ring and dimple but do not have an asymmetric mass. These results support models in which the F0 complex contains a ring of 9–12 c subunits with the b subunits located outside this ring, and show that scanning force microscopy is able to provide structural information on membrane proteins of molecular mass less than 200 000 Da.

Coupling between catalytic sites and the proton channel in F1F0-type ATPases

F1F0-type ATPases catalyse both ATP-driven proton translocation and proton-gradient-driven ATP synthesis. Recent cryoelectronmicroscopy and low-resolution X-ray studies provide a first glimpse at the structure of this complicated membrane-bound enzyme. The F1 part is roughly globular and linked to the membrane-intercalated F0 part by a narrow stalk domain, which contains the gamma-, delta- and epsilon-subunits along with domains of the b-subunit of the F0 part. Here, we review evidence that conformational and positional changes in the gamma- and epsilon-subunits provide the coupling between catalytic sites and proton translocation within the F1F0 complex.

H+/ATP ratio of proton transport-coupled ATP synthesis and hydrolysis catalysed by CF0F1–liposomes

The H+/ATP ratio and the standard Gibbs free energy of ATP synthesis were determined with a new method using a chemiosmotic model system. The purified H+‐translocating ATP synthase from chloroplasts was reconstituted into phosphatidylcholine/phosphatidic acid liposomes. During reconstitution, the internal phase was equilibrated with the reconstitution medium, and thereby the pH of the internal liposomal phase, pHin, could be measured with a conventional glass electrode. The rates of ATP synthesis and hydrolysis were measured with the luciferin/luciferase assay after an acid—base transition at different [ATP]/([ADP][Pi]) ratios as a function of ΔpH, analysing the range from the ATP synthesis to the ATP hydrolysis direction and the ΔpH at equilibrium, ΔpH (eq) (zero net rate), was determined. The analysis of the [ATP]/([ADP][Pi]) ratio as a function of ΔpH (eq) and of the transmembrane electrochemical potential difference, Δμ̃H+ (eq), resulted in H+/ATP ratios of 3.9 ± 0.2 at pH 8.45 and 4.0 ± 0.3 at pH 8.05. The standard Gibbs free energies of ATP synthesis were determined to be 37 ± 2 kJ/mol at pH 8.45 and 36 ± 3 kJ/mol at pH 8.05.

The thermodynamic H+/ATP ratios of the H+-ATPsynthases from chloroplasts and Escherichia coli

The H+/ATP ratio is an important parameter for the energy balance of all cells and for the coupling mechanism between proton transport and ATP synthesis. A straightforward interpretation of rotational catalysis predicts that the H+/ATP coincides with the ratio of the c-subunits to β-subunits, implying that, for the chloroplast and Escherichia coli ATPsynthases, numbers of 4.7 and 3.3 are expected. Here, the energetics described by the chemiosmotic theory was used to determine the H+/ATP ratio for the two enzymes. The isolated complexes were reconstituted into liposomes, and parallel measurements were performed under identical conditions. The internal phase of the liposomes was equilibrated with the acidic medium during reconstitution, allowing to measure the internal pH with a glass electrode. An acid–base transition was carried out and the initial rates of ATP synthesis or ATP hydrolysis were measured with luciferin/luciferase as a function of ΔpH at constant Q = [ATP]/([ADP][Pi]). From the shift of the equilibrium ΔpH as a function of Q the standard Gibbs free energy for phosphorylation, ΔGp0′; and the H+/ATP ratio were determined. It resulted ΔGp0′ = 38 ± 3 kJ·mol−1 and H+/ATP = 4.0 ± 0.2 for the chloroplast and H+/ATP = 4.0 ± 0.3 for the E. coli enzyme, indicating that the thermodynamic H+/ATP ratio is the same for both enzymes and that it is different from the subunit stoichiometric ratio.

CONFORMATIONAL CHANGES OF THE H+-ATPASE FROM ESCHERICHIA COLI UPON NUCLEOTIDE BINDING DETECTED BY SINGLE MOLECULE FLUORESCENCE

Using a confocal fluorescence microscope with an avalanche photodiode as detector, we studied the fluorescence of the tetramethylrhodamine labeled F1 part of the H+‐ATPase from Escherichia coli, EF1, carrying the γT106‐C mutation [Aggeler, J.A. and Capaldi, R.A. (1992) J. Biol. Chem. 267, 21355–21359] in aqueous solution upon excitation with a mode‐locked argon ion laser at 528 nm. The diffusion of the labeled EF1 through the confocal volume gives rise to photon bursts, which were analyzed with fluorescence correlation spectroscopy, resulting in a diffusion coefficient of 3.3×10−7 cm2 s−1. In the presence of nucleotides the diffusion coefficient increases by about 15%. This effect indicates a change of the shape and/or the volume of the enzyme upon binding of nucleotides, i.e. fluorescence correlation spectroscopy with single EF1 molecules allows the detection of conformational changes.

The Activity of the ATP Synthase from Escherichia coli Is Regulated by the Transmembrane Proton Motive Force

The ATP synthase from Escherichia coli was reconstituted into liposomes from phosphatidylcholine/phosphatidic acid. The proteoliposomes were energized by an acid-base transition and a K+/valinomycin diffusion potential, and one second after energization, the electrochemical proton gradient was dissipated by uncouplers, and the ATP hydrolysis measurement was started. In the presence of ADP and Pi, the initial rate of ATP hydrolysis was up to 9-fold higher with pre-energized proteoliposomes than with proteoliposomes that had not seen an electrochemical proton gradient. After dissipating the electrochemical proton gradient, the high rate of ATP hydrolysis decayed to the rate without pre-energization within about 15 s. During this decay the enzyme carried out approximately 100 turnovers. In the absence of ADP and Pi, the rate of ATP hydrolysis was already high and could not be significantly increased by pre-energization. It is concluded that ATP hydrolysis is inhibited when ADP and Pi are bound to the enzyme and that a high Δμ̃H+ is required to release ADP and Pi and to convert the enzyme into a high activity state. This high activity state is metastable and decays slowly when Δμ̃H+ is abolished. Thus, the proton motive force does not only supply energy for ATP synthesis but also regulates the fraction of active enzymes.

Comparison of the H+/ATP ratios of the H+-ATP synthases from yeast and from chloroplast.

F0F1-ATP synthases use the free energy derived from a transmembrane proton transport to synthesize ATP from ADP and inorganic phosphate. The number of protons translocated per ATP (H+/ATP ratio) is an important parameter for the mechanism of the enzyme and for energy transduction in cells. Current models of rotational catalysis predict that the H+/ATP ratio is identical to the stoichiometric ratio of c-subunits to β-subunits. We measured in parallel the H+/ATP ratios at equilibrium of purified F0F1s from yeast mitochondria (c/β = 3.3) and from spinach chloroplasts (c/β = 4.7). The isolated enzymes were reconstituted into liposomes and, after energization of the proteoliposomes with acid–base transitions, the initial rates of ATP synthesis and hydrolysis were measured as a function of ΔpH. The equilibrium ΔpH was obtained by interpolation, and from its dependency on the stoichiometric ratio, [ATP]/([ADP]·[Pi]), finally the thermodynamic H+/ATP ratios were obtained: 2.9 ± 0.2 for the mitochondrial enzyme and 3.9 ± 0.3 for the chloroplast enzyme. The data show that the thermodynamic H+/ATP ratio depends on the stoichiometry of the c-subunit, although it is not identical to the c/β ratio.

Proton transport coupled ATP synthesis by the purified yeast H+-ATP synthase in proteoliposomes

The H(+)/ATP synthase from yeast mitochondria, MF₀F₁, was purified and reconstituted into liposomes prepared from phosphatidylcholine and phosphatidic acid. Analysis by mass spectrometry revealed the presence of all subunits of the yeast enzyme with the exception of the K-subunit. The MF₀F₁ liposomes were energized by acid-base transitions (DeltapH) and a K(+)/valinomycin diffusion potential (Deltaphi). ATP synthesis was completely abolished by the addition of uncouplers as well as by the inhibitor oligomycin. The rate of ATP synthesis was optimized as a function of various parameters and reached a maximum value (turnover number) of 120s⁻¹ at a transmembrane pH difference of 3.2 units (at pH(in)=4.8 and pH(out)=8.0) and a Deltaphi of 133mV (Nernst potential). Functional studies showed that the monomeric MF₀F₁, was fully active in ATP synthesis. The turnover increased in a sigmoidal way with increasing internal and decreasing external proton concentration. The dependence of the turnover on the phosphate concentration and the dependence of K(M) on pH(out) indicated that the substrate for ATP synthesis is the monoanionic phosphate species H₂PO⁻₄.

Intrinsic uncoupling in the ATP synthase of Escherichia coli

The ATP hydrolysis activity and proton pumping of the ATP synthase of Escherichia coli in isolated native membranes have been measured and compared as a function of ADP and Pi concentration. The ATP hydrolysis activity was inhibited by Pi with an half-maximal effect at 140 microM, which increased progressively up in the millimolar range when the ADP concentration was progressively decreased by increasing amounts of an ADP trap. In addition, the relative extent of this inhibition decreased with decreasing ADP. The half-maximal inhibition by ADP was found in the submicromolar range, and the extent of inhibition was enhanced by the presence of Pi. The parallel measurement of ATP hydrolysis activity and proton pumping indicated that, while the rate of ATP hydrolysis was decreased as a function of either ligand, the rate of proton pumping increased. The latter showed a biphasic response to the concentration of Pi, in which an inhibition followed the initial stimulation. Similarly as previously found for the ATP synthase from Rhodobacter caspulatus [P. Turina, D. Giovannini, F. Gubellini, B.A. Melandri, Physiological ligands ADP and Pi modulate the degree of intrinsic coupling in the ATP synthase of the photosynthetic bacterium Rhodobacter capsulatus, Biochemistry 43 (2004) 11126-11134], these data indicate that the E. coli ATP synthase can operate at different degrees of energetic coupling between hydrolysis and proton transport, which are modulated by ADP and Pi.

Physiological Ligands ADP and Pi Modulate the Degree of Intrinsic Coupling in the ATP Synthase of the Photosynthetic Bacterium Rhodobacter capsulatus

The proton-pumping and the ATP hydrolysis activities of the ATP synthase of Rhodobacter capsulatus have been compared as a function of the ADP and P(i) concentrations. The proton pumping was measured either with the transmembrane pH difference probe, 9-amino-6-chloro-2-methoxyacridine, or with the transmembrane electric potential difference probe, bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol, obtaining consistent results. The comparison indicates that an intrinsic uncoupling of ATP synthase is induced when the concentration of either ligand is decreased. The half-maximal effect was found in the submicromolar range for ADP and at about 70 microM for P(i). It is proposed that a switch from a partially uncoupled state of ATP synthase to the coupled state is induced by the simultaneous binding of ADP and P(i).