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 puri®ed 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 DpH, analysing the range from the ATP synthesis to the ATP hydrolysis direction and the DpH at equilibrium, DpH (eq) (zero net rate), was determined. The analysis of the [ATP]/ ([ADP][P i ]) ratio as a function of DpH (eq) and of the transmembrane electrochemical potential difference, Dm H + (eq), resulted in H + /ATP ratios of 3.9 6 0.2 at pH 8.45 and 4.0 6 0.3 at pH 8.05. The standard Gibbs free energies of ATP synthesis were determined to be 37 6 2 kJ/mol at pH 8.45 and 36 6 3 kJ/mol at pH 8.05.

Comparison of the H + /ATP ratios of the H + -ATP synthases from yeast and from chloroplast

“…When the ΔpH was abolished by addition of 10 μM nigericin, CF 0 F 1 and MF 0 F 1 showed different responses: CF 0 F 1 did not show any ATP hydrolysis, whereas MF 0 F 1 did. This is in agreement with previous results (18,19) and confirms that CF 0 F 1 is a strongly regulated enzyme, which is inactive in the absence of a pre-energization by Δμ H þ (23). We concluded that CF 0 F 1 did not catalyze uncoupled ATP hydrolysis and that, therefore, no corrections for uncoupled ATP hydrolysis were required.…”

“…To apply this method we constructed a minimal chemiosmotic system (18,19). Liposomes from phosphatidylcholine/phosphatidic acid were prepared, and either the chloroplast or the mitochondrial enzyme was reconstituted into the liposome membrane.…”

“…At the mixing time (t = 0), a transmembrane ΔpH is established, which decays thereafter within a few minutes owing to passive and phosphorylating proton effluxes. Because pH in and pH out were measured with the same calibrated glass electrode, both parameters were known with high accuracy and precision, the error in the initial ΔpH measurement being smaller than 0.02 units (18). With the glass electrode the proton activities are detected, so that no further corrections were necessary for determination of ΔpH values.…”

“…Hence, their respective H + /ATP ratios, based on the assumptions mentioned above, should be 3.3 and 4.7. We have isolated the two complexes and reconstituted them into liposomes, which, if subjected to acid-base transitions to generate a high protonmotive force, were able to synthesize ATP at physiological rates (17,18). The technique of acid-base transitions offers the great advantages of (i) measuring the imposed transmembrane ΔpH with the accuracy of the pH electrode, thus making high-precision quantitative studies possible (18)(19)(20), and (ii) allowing the testing of ATP synthases from different species with the same method and under identical experimental conditions.…”

Rotary Movements within the ATP Synthase do not Constitute an Obligatory Element of the Catalytic Mechanism

“…If the reported number of c subunits in the chloroplast enzyme (52) is correct, the proton/ATP stoicheiometry should be 4.67 (14 divided by 3) according to the current rotational model (rotation of the ring of c subunits is coupled with the passage of one proton through each of the c subunits and with rotation of the g subunit without slip), while this number has been determined as being 4.0 (53,54). A movement over 120 degrees, however, can accommodate this number quite well.…”