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 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][P _i ]). From the shift of the equilibrium ΔpH as a function of Q the standard Gibbs free energy for phosphorylation, Δ G _p ⁰ ′; and the H ⁺ /ATP ratio were determined. It resulted Δ G _p ⁰ ′ = 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.

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.

F ₀ F ₁ -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 F ₀ F ₁ s 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]·[P _i ]), 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.