Peter Gräber

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.

H⁺‐ATP synthases are molecular machines which couple transmembrane proton transport with ATP synthesis from ADP and inorganic phosphate by a rotational mechanism. Single‐pair fluorescence resonance energy transfer (spFRET) in single molecules is a powerful tool to analyse conformational changes. It is used to investigate subunit movements in H⁺‐ATP synthases from E. coli (EF₀F₁) and from spinach chloroplasts (CF₀F₁) during catalysis. The enzymes are incorporated into liposome membranes, and this allows the generation of a transmembrane pH difference, which is necessary for ATP synthesis. After labelling of appropriate sites on different subunits with fluorescence donor and acceptor, the kinetics of spFRET are measured. Analysis of the E_FRET traces reveals rotational movement of the ε and γ subunits in 120° steps with opposite directions during ATP synthesis and ATP hydrolysis. The stepped movement is characterized by a 120° step faster than 1 ms followed by a rest period with an average dwell time of 15 ms, which is in accordance with the turnover time of the enzyme. In addition to the three conformational states during catalysis, also an inactive conformation is found, which is observed after catalysis.

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.