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 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.