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

Abstract

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, pH(in), 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][P(i)]) ratios as a function of deltapH, analysing the range from the ATP synthesis to the ATP hydrolysis direction and the deltapH at equilibrium, deltapH (eq) (zero net rate), was determined. The analysis of the [ATP]/([ADP][P(i)]) ratio as a function of deltapH (eq) and of the transmembrane electrochemical potential difference, delta micro approximately (H)(+) (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.

Fig. 1. Scheme of the chemiosmotic system. Liposomes were made from phosphatidylcholine and contained 5 mol% phosphatidic acid. Their mean diameter was 120 nm, containing 1.3 × 10⁵ lipid molecules and one CF₀F₁. Each liposome contained ∼800 valinomycin molecules.

Fig. 2. ATP synthesis and ATP hydrolysis after generation of a transmembrane ΔpH. Proteoliposomes were incubated in the acidic medium containing luciferin/luciferase for 30 s and the baseline was registered. The acid–base transition was carried out in the luminometer by addition of 250 µl of basic medium. The final pH_out was always 8.45 and the final CF₀F₁ concentration 7.6 nM. This addition gives rise to an immediate change in the baseline. For clarity, the baseline was shifted to the luminescence level obtained in the absence of any reaction. This step is followed by a slow luminescence increase (ATP synthesis) or decrease (ATP hydrolysis). (A–D) refer to different stoichiometric ratios Q; the concentrations of substrates, products and Q are listed in Table II. The luminescence was calibrated by addition of standard ATP. The different ΔpH values are given next to each trace. The slopes at t = 0 give the initial rates in mol ATP per mol CF₀F₁ per s, i.e. in s^–1.

Fig. 3. Rate of ATP synthesis and ATP hydrolysis as a function of the transmembrane ΔpH at pH_out = 8.45. Each curve represents data at constant stoichiometric ratio Q: 0.805 (A), 4.65 (B), 28.7 (C) and 230 (D). Arrows indicate ΔpH_eq, where the rates of ATP synthesis and ATP hydrolysis are equal. Top: data from Figure 2. Bottom: the data from Figure 2 were corrected for activation as described in the text.

Fig. 4. Rate of ATP synthesis and ATP hydrolysis as a function of the transmembrane ΔpH at pH_out = 8.05. Each curve represents data at constant stoichiometric ratio Q: 0.07 (A), 0.52 (B), 2.07 (C) and 11.6 (D). Arrows indicate ΔpH_eq, where the rates of ATP synthesis and ATP hydrolysis are equal. Top: measured rates from experiments similar to that described in Figure 2. Bottom: the data from the top were corrected for activation as described in the text.

Fig. 5. Stoichiometric product [ATP]/([ADP] [P_i]) as a function of Δµ̃_H⁺ (eq). Data from Figures 3 and 4 and Table II were plotted according to equation 4. The slopes give the number n; the y-intercepts give the standard Gibbs free energies ΔG_p°′ at pH_out = 8.05 and pH_out = 8.45.