Modulation of proton pumping efficiency in bacterial ATP synthases

Abstract

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 (phospho-enol-pyruvate (PEP) and pyruvate kinase (PK)). The steady state values of DeltapH 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 approximately 0.5 microM was estimated. The extent of proton uptake was also strongly dependent on the addition of phosphate to the assay medium. The Kd for this effect was about 70 microM. 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 submillimolar 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 favors an inefficient (slipping) functional state. The switch between these states can probably be related to a structural change in the C-terminal alpha-helical hairpin of the epsilon-subunit, from an extended conformation, in which ATP hydrolysis is tightly coupled to proton pumping, to a retracted one, in which ATP hydrolysis and proton pumping are loosely coupled.

Regulation of the F1F0-ATP Synthase Rotary Nanomotor in its Monomeric-Bacterial and Dimeric-Mitochondrial Forms

“…It can also be questioned whether dimerization actually increases the coupling efficiency of the enzyme, given that the monomeric bacterial enzyme is already highly efficient as a coupling factor; indeed, the most efficient and practically unidirectional ATP synthases described so far are those of P. denitrificans [30] and of a thermoalkaliphilic bacterium [31]. However, it is also recalled that, in α-proteobacteria and even in eubacteria such as E. coli, it has been described that the rotary turnover of the F 1 portion undergoes slippage from the proton conduction through F 0 under conditions of low ADP and Pi concentrations [76,77]. This slipping has not been observed for the mitochondrial enzyme, probably because the rotor and stator interfaces of each monomer interact more efficiently in the dimeric or oligomeric forms of the enzyme.…”

Ethidium bromide efflux by Salmonella: modulation by metabolic energy, pH, ions and phenothiazines

“…Because ABC transporters hydrolyse ATP subsequent to their binding the substrate, reduction of ATP levels reduces efflux of the substrate. At pH 8 the hydrolysis of ATP by ATP synthase is favoured [22][23][24]. In order to determine whether the marked inhibition of efflux promoted by TZ at pH 8 is due to inhibition of an ABC transporter, the efflux assay was conducted in glucose-saline at pH 8 with increasing concentrations of OV.…”

Tethering polypeptides through bifunctional PEG cross‐linking agents to probe protein function: Application to ATP synthase

“…It will be interesting to see if direct observation of this slippage may be possible using single molecule techniques 57, 58. The conformation of the C‐terminal domain has been shown to change in response to different nucleotides59 and a regulatory role has been proposed 59–61…”