ATP hydrolysis in ATP synthases can be differently coupled to proton transport and modulated by ADP and phosphate: a structure based model of the mechanism.

In the ATP synthases of Escherichia coli ADP and phosphate exert an apparent regulatory role on the efficiency of proton transport coupled to the hydrolysis of ATP. Both molecules induce clearly biphasic effects on hydrolysis and proton transfer. At intermediate concentrations (approximately 0.5-1 microM and higher) ADP inhibits hydrolysis and proton transfer; a quantitative analysis of the fluxes however proves that the coupling efficiency remains constant in this concentration range. On the other hand at nanomolar concentrations of ADP (a level obtainable only using an enzymatic ATP regenerating system) the efficiency of proton transport drops progressively, while the rate of hydrolysis remains high. Phosphate, at concentrations>or=0.1 mM, inhibits hydrolysis only if ADP is present at sufficiently high concentrations, keeping the coupling efficiency constant. At lower ADP levels phosphate is, however, necessary for an efficiently coupled catalytic cycle. We present a model for a catalytic cycle of ATP hydrolysis uncoupled from the transport of protons. The model is based on the available structures of bovine and yeast F1 and on the known binding affinities for ADP and Pi of the catalytic sites in their different functional states. The binding site related to the inhibitory effects of Pi (in association with ADP) is identified as the alphaHCbetaHC site, the pre-release site for the hydrolysis products. We suggest, moreover, that the high affinity site, associated with the operation of an efficient proton transport, could coincide with a conformational state intermediate between the alphaTPbetaTP and the alphaDPbetaDP (similar to the transition state of the hydrolysis/synthesis reaction) that does not strongly bind the ligands and can exchange them rather freely with the external medium. The emptying of this site can lead to an unproductive hydrolysis cycle that occurs without a net rotation of the central stalk and, consequently, does not translocate protons

Intrinsic uncoupling in the ATP synthase of Escherichia coli.

The ATP hydrolysis activity and proton pumping of the ATP synthase of Escherichia coli in isolated native membranes have been measured and compared as a function of ADP and Pi concentration. The ATP hydrolysis activity was inhibited by Pi with an half-maximal effect at 140 muM, which increased progressively up in the millimolar range when the ADP concentration was progressively decreased by increasing amounts of an ADP trap. In addition, the relative extent of this inhibition decreased with decreasing ADP. The half-maximal inhibition by ADP was found in the submicromolar range, and the extent of inhibition was enhanced by the presence of Pi. The parallel measurement of ATP hydrolysis activity and proton pumping indicated that, while the rate of ATP hydrolysis was decreased as a function of either ligand, the rate of proton pumping increased. The latter showed a biphasic response to the concentration of Pi, in which an inhibition followed the initial stimulation. Similarly as previously found for the ATP synthase from Rhodobacter caspulatus [P. Turina, D. Giovannini, F. Gubellini, B.A. Melandri, Physiological ligands ADP and Pi modulate the degree of intrinsic coupling in the ATP synthase of the photosynthetic bacterium Rhodobacter capsulatus, Biochemistry 43 (2004) 11126-11134], these data indicate that the E. coli ATP synthase can operate at different degrees of energetic coupling between hydrolysis and proton transport, which are modulated by ADP and Pi

Effect of Pi and ADP on the intrinsic uncoupling in the isolated and reconstituted ATPsynthase of E-coli

We have recently shown that the H+/ATP ratio can significantly decrease during ATP hydrolysis by the ATPsynthase of Rb. capsulatus, when the concentration of either ADP or Pi is maintained at a low level. This same phenomenon has then been observed in isolated membranes of E. coli. We have now purified the ATPsynthase of E. coli and reconstituted it into liposomes, in order to verify whether the same behavior could be observed in the isolated enzyme. The ATP hydrolysis and proton pumping activity were measured under the same experimental conditions. The hydrolysis was measured either with the colorimetric pH indicator Phenol Red or with an ATP regenerating enzymatic assay, and the proton pumping was evaluated by a calibrated ACMA assay. The hydrolysis activity was inhibited by Pi with an apparent Kd of 400 μM, while the steady state ΔpH was stimulated up to 200 μM Pi and was only slightly inhibited up to 1000 μM Pi. Both the inhibition of ATP hydrolysis and the stimulation of proton pumping as a function of Pi were lost upon ADP removal by an ADP trap. We conclude that the isolated and reconstituted ATPsynthase of E. coli can vary its degree of coupling as a function of Pi and ADP

Effect of Pi and ADP on the intrinsic uncoupling in the isolated and reconstituted ATPsynthase of E-coli

We have recently shown that the H+/ATP ratio can significantly decrease during ATP hydrolysis by the ATPsynthase of Rb. capsulatus, when the concentration of either ADP or Pi is maintained at a low level. This same phenomenon has then been observed in isolated membranes of E. coli. We have now purified the ATPsynthase of E. coli and reconstituted it into liposomes, in order to verify whether the same behavior could be observed in the isolated enzyme. The ATP hydrolysis and proton pumping activity were measured under the same experimental conditions. The hydrolysis was measured either with the colorimetric pH indicator Phenol Red or with an ATP regenerating enzymatic assay, and the proton pumping was evaluated by a calibrated ACMA assay. The hydrolysis activity was inhibited by Pi with an apparent Kd of 400 μM, while the steady state ΔpH was stimulated up to 200 μM Pi and was only slightly inhibited up to 1000 μM Pi. Both the inhibition of ATP hydrolysis and the stimulation of proton pumping as a function of Pi were lost upon ADP removal by an ADP trap. We conclude that the isolated and reconstituted ATPsynthase of E. coli can vary its degree of coupling as a function of Pi and ADP

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

Role of the epsilon subunit C-terminal domain of the Escherichia coli ATP synthase in modulating activity and coupling degree

The \u3b5-subunit of the ATP-synthase is known as an endogenous inhibitor of the hydrolysis activity of the complex and its \u3b1-helical C-terminal domain undergoes drastic conformational changes between a non-inhibited form (down-state) and an inhibited form (up-state). Even though this C-terminal domain does not appear to be essential for ATP synthesis activity, there are evidence of its involvement in the coupling mechanism of the pump [1]. Recently, it has been proposed that the coupling degree of the ATP-synthase can vary as a function of ADP and Pi concentration [2-4]. In the present work we explored the possible role of the C-terminal domain in this ligand-dependent uncoupling, by examining a C-terminally truncated e mutant of E.coli. We have developed a low copy number expression vector carrying an extra copy of uncC with the aim of promoting normal levels of assembly of the mutated ATP-synthase complex. Both the wild-type and the \u3b588-stop truncated strains showed well energized membranes. Noticeably, they showed a marked difference in their response to Pi: the Pi-induced inhibition of membrane-bound ATPase activity appeared to be completely lost in the truncated mutant, and the Pi-induced coupling increase was very reduced. However, pre-incubation of the mutated enzyme with ADP at rather low concentrations ([ADP] = 100 nM) largely restored the Pi-induced hydrolysis inhibition. Analogously, the increase in coupling degree induced by Pi was resumed after incubation with extremely low [ADP] (1 nM). This suggests that, contrary to wild-type, the truncated mutant had lost its bound ADP, most likely during membrane preparation, as a consequence of a lower affinity for ADP. The whole set of data is interpreted to indicate that, in the wild-type ATP-synthase, one ADP-binding site at very high affinity (Kd < 1 nM) mainly influences the coupling degree, and one ADP-binding site atintermediate affinity mainly inhibits the hydrolytic activity. The \u3b5-subunit C-terminal domain appears to increase the affinity of these two ADP binding sites, thus playing a major role in modulating both the activity and coupling degree of the ATP-synthase

Modulation of coupling in the Escherichia coli ATP synthase by ADP and Pi: Role of the ε subunit C-terminal domain

The ε-subunit of ATP-synthase is an endogenous inhibitor of the hydrolysis activity of the complex and its α-helical C-terminal domain (εCTD) undergoes drastic changes among at least two different conformations. Even though this domain is not essential for ATP synthesis activity, there is evidence for its involvement in the coupling mechanism of the pump. Recently, it was proposed that coupling of the ATP synthase can vary as a function of ADP and Pi concentration. In the present work, we have explored the possible role of the εCTD in this ADP- and Pi-dependent coupling, by examining an εCTD-lacking mutant of Escherichia coli. We show that the loss of Pi-dependent coupling can be observed also in the εCTD-less mutant, but the effects of Pi on both proton pumping and ATP hydrolysis were much weaker in the mutant than in the wild-type. We also show that the εCTD strongly influences the binding of ADP to a very tight binding site (half-maximal effect ≈ 1 nM); binding at this site induces higher coupling in EFOF1 and increases responses to Pi. It is proposed that one physiological role of the εCTD is to regulate the kinetics and affinity of ADP/Pi binding, promoting ADP/Pi-dependent coupling

The ATP Synthase atpHAGDC (F1) Operon from Rhodobacter capsulatus

The atpHAGDC operon of Rhodobacter capsulatus, containing the five genes coding for the F(1) sector of the ATP synthase, has been cloned and sequenced. The promoter region has been defined by primer extension analysis. It was not possible to obtain viable cells carrying atp deletions in the R. capsulatus chromosome, indicating that genes coding for ATP synthase are essential, at least under the growth conditions tested. We were able to circumvent this problem by combining gene transfer agent transduction with conjugation. This method represents an easy way to construct strains carrying mutations in indispensable genes