INTRINSIC UNCOUPLING IN THE ATP SYNTHASE OF E.COLI

The H+/ATP ratio in the catalysis of ATP synthase has generally been considered a fixed parameter. However, in the ATP synthase of the photosynthetic bacterium Rhodobacter capsulatus, we have recently shown that this ratio can significantly decrease during ATP hydrolysis when the concentration of either ADP or Pi is maintained at a low level (1). We have next focused our attention on the ATP synthase of E.coli, looking specifically for evidence of intrinsic uncoupling in this organism as well. The hydrolysis activity of the purified, reconstituted E.coli enzyme has been shown to be strongly inhibited, in the presence of ADP, by Pi, with an apparent Kd in the order of 500 microM, and by ADP, in the microM range (2). We have reproduced these results measuring as a function of Pi and ADP the DCCD-sensitive ATP hydrolysis activity of E.coli internal membranes. In contrast to this monotonic inhibition, however, the proton pumping activity of the enzyme, as estimated under the same experimental conditions by the fluorescence quenching of the DeltapH-sensitive probe ACMA, showed a clearly biphasic progression, both for Pi, increasing from 0 up to approximately 200 microM, and for ADP, increasing from 0 to few microM. This result can only be explained if the occupancy of ADP and Pi binding sites shifts the enzyme from a (partially) uncoupled state to a normally coupled state. We conclude that the phenomenon of “intrinsic uncoupling”, first shown in the ATP synthase of Rb. capsulatus, also takes place in the E.coli enzyme, suggesting its likey occurrence in all Prokaryotes. (1) Turina P., Giovannini D., Gubellini F. and Melandri B.A. (2004) Biochemistry 43: 11126-34. (2) Fischer S., Gräber P. and Turina P. (2000) J. Biol. Chem. 275: 30157-62

MODULATION OF PROTON PUMPING EFFICIENCY IN BACTERIAL ATP SYNTHASES

The ATP synthase in chromatophores of Rhodobacter caspulatus can effectively generate a transmembrane pH difference coupled to the hydrolysis of ATP. The rate of hydrolysis was rather insensitive to the depletion of ADP in the assay medium by an ATP regenerating system (phosphoenolpyruvate (PEP) and pyruvate kinase (PK)). The steady state values of ΔpH were however drastically reduced as a consequence of ADP depletion. The clamped concentrations of ADP obtained using different PK activities in the assay medium could be calculated and an apparent Kd ≈ 0.5 μM was estimated. The extent of proton uptake was also strongly dependent on the addition of phosphate (Pi) to the assay medium. The Kd for this effect was about 70 μM. Analogous experiments were performed in membrane fragment from Escherichia coli. In this case, however, the hydrolysis rate was strongly inhibited by Pi, added up to 3 mM. Inhibition by Pi was nearly completely suppressed following depletion of ADP. The Kd’s for the ADP and Pi were in the micromolar range and submillimoar range respectively and were mutually dependent from the concentration of the other ligand. Contrary to hydrolysis, the pumping of protons was rather insensitive to changes in the concentrations of the two ligands. At intermediate concentrations, proton pumping was actually stimulated, while the hydrolysis was inhibited. It is concluded that, in these two bacterial organisms, ADP and phosphate induce a functional state of the ATP synthase competent for a tightly coupled proton pumping, while the depletion of either one of these two ligands favours an inefficient (slipping) functional state. The switch between these states can probably be related to the structural change in the C-terminal α-helical hairpin of the ε-subunit, from the extended conformation, in which ATP hydrolysis is tightly coupled to proton pumping, to the retracted conformation, in which ATP hydrolysis and proton pumping are loosely coupled

A single-point mutation in the ATP synthase of Rb. capsulatus impairing the stability of the protonmotive forceactivated state

The single-point mutation gammaM23-K introduced in the ATP synthase of E. coli has been reported to perturb the coupling efficiency between ATP hydrolysis and proton pumping as measured with the ACMA assay (1). Supporting this conclusion, the ATP synthesis rate was more affected compared to wild-type than the ATP hydrolysis rate, by about threefold (2). In addition, a study of interaction between subunits indicated that, in the mutated complex, the epsilon subunit inhibition of ATPase activity was not relieved upon binding of F1 to the membrane as observed in the wild-type (2). With the aim of further investigating the uncoupling process in a photosynthetic system in which analysis of the kinetics of the phosphorylating proton fluxes is possible (3), we have introduced this same mutation in the ATP synthase of Rb. capsulatus.In this organism, ATP synthesis and hydrolysis rates were impaired to a similar extent, both to approximately 1/3 of wild-type. Analysis of phosphorylating proton fluxes and associated ATP synthesis in the mutated and wild-type enzymes has not revealed uncoupling.However, the protonmotive force-activated state (measured as the transient increase of the ATP hydrolysis rate upon addition of uncouplers to energised vesicles), decayed extremely fast compared to wild-type. In agreement with this finding, the coupled proton flux through FoF1 induced by a single flash, which is usually observed in the wild-type enzyme in the presence of ADP and Pi, was completely absent. We conclude that the gammaM23-K mutated ATP synthase of Rb. capsulatus is an excellent system for studying the mechanism of ATP synthase activation by the protonmotive force. References [1] K. Shin, R.K. Nakamoto, M. Maeda, M. Futai, J. Biol Chem. 267 (1992) 20835–20839. [2] M.K. Al-Shawi, C.J. Ketchum, R.K. Nakamoto, J. Biol Chem. 272 (1997) 2300–2306. [3] B.A. Feniouk, D.A. Cherepanov, W. Junge, A.Y. Mulkidjanian, Biochim. Biophys. Acta 506 (2001) 189–203

Met23Lys mutation in subunit gamma of F(O)F(1)-ATP synthase from Rhodobacter capsulatus impairs the activation of ATP hydrolysis by protonmotive force.

Abstract H+-FOF1-ATP synthase couples proton flow through its membrane portion, FO, to the synthesis of ATP in its headpiece, F1. Upon reversal of the reaction the enzyme functions as a proton pumping ATPase. Even in the simplest bacterial enzyme the ATPase activity is regulated by several mechanisms, involving inhibition by MgADP, conformational transitions of the ε subunit, and activation by protonmotive force. Here we report that the Met23Lys mutation in the γ subunit of the Rhodobacter capsulatus ATP synthase significantly impaired the activation of ATP hydrolysis by protonmotive force. The impairment in the mutant was due to faster enzyme deactivation that was particularly evident at low ATP/ADP ratio. We suggest that the electrostatic interaction of the introduced γLys23 with the DELSEED region of subunit β stabilized the ADP-inhibited state of the enzyme by hindering the rotation of subunit γ rotation which is necessary for the activation