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.