The Activity of the ATP Synthase from Escherichia coli Is Regulated by the Transmembrane Proton Motive Force

Susanne Fischer,Peter Gräber,Paola Turina

doi:10.1074/jbc.275.39.30157

Susanne Fischer, Peter Gräber + Show 1 more

Open Access

https://doi.org/10.1074/jbc.275.39.30157

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Abstract

The ATP synthase from Escherichia coli was reconstituted into liposomes from phosphatidylcholine/phosphatidic acid. The proteoliposomes were energized by an acid-base transition and a K(+)/valinomycin diffusion potential, and one second after energization, the electrochemical proton gradient was dissipated by uncouplers, and the ATP hydrolysis measurement was started. In the presence of ADP and P(i), the initial rate of ATP hydrolysis was up to 9-fold higher with pre-energized proteoliposomes than with proteoliposomes that had not seen an electrochemical proton gradient. After dissipating the electrochemical proton gradient, the high rate of ATP hydrolysis decayed to the rate without pre-energization within about 15 s. During this decay the enzyme carried out approximately 100 turnovers. In the absence of ADP and P(i), the rate of ATP hydrolysis was already high and could not be significantly increased by pre-energization. It is concluded that ATP hydrolysis is inhibited when ADP and P(i) are bound to the enzyme and that a high Delta mu(H(+)) is required to release ADP and P(i) and to convert the enzyme into a high activity state. This high activity state is metastable and decays slowly when Delta mu(H(+)) is abolished. Thus, the proton motive force does not only supply energy for ATP synthesis but also regulates the fraction of active enzymes.

Highlights

The ATP synthase from Escherichia coli was reconstituted into liposomes from phosphatidylcholine/phosphatidic acid
The proteoliposomes were energized by an acid-base transition and a K؉/valinomycin diffusion potential, and one second after energization, the electrochemical proton gradient was dissipated by uncouplers, and the ATP hydrolysis measurement was started
Chromatophores, and some bacterial F0F1-ATPases, it appears that ⌬pH and ⌬␸ are kinetically equivalent, i.e. the rate of ATP synthesis is changed by the same factor when either ⌬␸ is changed by 59 mV or ⌬pH is changed by one unit (14 –17)

Summary

EXPERIMENTAL PROCEDURES

Purification and Reconstitution of EF0F1 into Liposomes—E. coli strain DK8 (⌬unc) transformed with plasmid pBWU13 [32] was a gift from Prof. The proteoliposomes were obtained in a buffer at pH 8.0 containing 13 mM Tricine, 8 mM succinate, 9 mM NaOH, 0.5 mM KCl, and 4 mM MgCl2 with a lipid concentration of approximately 8 g/liter and a EF0F1 concentration of 30 nM. For energization at lower ⌬␸ or in the absence of ⌬␸, KCl was added to final concentrations of 8 and 100 mM, respectively, and the proteoliposomes were incubated at room temperature for 4 h in the presence of 50 ␮M valinomycin prior to measurements.

The abbreviations used are

RESULTS

DISCUSSION