Comparison of ΔpH- and Δφ-driven ATP synthesis catalyzed by the H+-ATPases from Escherichia coli or chloroplasts reconstituted into liposomes

AbstractThe H+-ATPases from Escherichia coli, EF0F1, and from chloroplasts, CF0F1, were reconstituted in liposomes from phosphatidylcholine/phosphatidic acid. The proteoliposomes were energized by an acid-base transition and a K+/valinomycin diffusion potential and the initial rate of ATP synthesis was measured as a function of the transmembrane pH difference, ΔpH, and the electric potential difference, Δφ. With EF0F1, a rate of 80 s−1 is observed at ΔpH=4.1 and Δφ≈140 mV. The rate decreases sigmoidally with Δφ and at Δφ≈0 mV, the rate is about 1 s−1 although ΔpH is still 4.1. Under the same conditions with CF0F1, a rate of 280 s−1 is observed which decreases to 190 s−1 when Δφ is abolished, i.e. ATP synthesis catalyzed by EF0F1 and CF0F1 depends in a different way on ΔpH and Δφ. EF0F1-catalyzed ATP synthesis was measured as a function of ΔpH at a constant Δφ. The rate depends sigmoidally on ΔpH reaching a maximal rate which cannot be further increased by increasing ΔpH. However, this maximal rate depends on Δφ, i.e. ΔpH and Δφ are not kinetically equivalent in driving ATP synthesis. We assume that EF0F1 must be converted into a metastable, active state before it catalyzes proton transport-coupled ATP synthesis. For EF0F1, this activation step depends only on Δφ, whereas for CF0F1, the activation depends on ΔpH and Δφ