Quantitative evaluation of the intrinsic uncoupling modulated by ADP and Pi in the reconstituted ATP synthase of Escherichia coli.

The ATP hydrolysis activity and the proton pumping activity of the isolated and reconstituted ATP synthase of Escherichia coli, the latter evaluated from the ACMA fluorescence quenching and its calibration by means of acid-base transitions, have been measured under the same experimental conditions as a function of Pi and ADP concentration. Pi monotonically inhibited the ATP hydrolysis rate with a half-maximal effect at 510 µM, whereas it stimulated proton pumping up to 100-200 µM, inhibiting it only at higher concentrations. Progressively decreasing the steady state ADP concentration during hydrolysis by means of increasing PK activities, working as an ADP trap, down to estimated concentrations in the range of few tens of nanomolar, led first to an increase of the hydrolytic rate, which was however unaffected in the lower concentration range. In parallel, a concomitant decrease of the proton pumping activity was observed, most evident in this lower ADP concentration range. We explain these data by the presence of two ADP and Pi binding sites, one of which featuring a very high affinity for ADP (estimated Kd in the tens of nanomolar range), and mainly involved in the observed change in the efficiency of proton pumping, the second site, of lower affinity for ADP, mainly involved in the inhibition of ATP hydrolysis and proton pumping alike. The quantitative analysis shows that the efficiency of proton pumping, i.e. the effective number of translocated protons per hydrolyzed ATP (coupling ratio), can drop down to at least 15% relative to that of the fully coupled enzyme