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.

MODULATION OF PROTON PUMPING EFFICIENCY IN BACTERIAL ATP SYNTHASES / P. Turina; A. Rebecchi; M. D'Alessandro; S. Anefors; B.A. Melandri. - In: BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS. - ISSN 0005-2728. - STAMPA. - 1757:5–6, Supplement(2006), pp. 41-41. (Intervento presentato al convegno 14th European Bioenergetic Conference tenutosi a Moscow, RUSSIA nel JUL 22-27, 2006).

MODULATION OF PROTON PUMPING EFFICIENCY IN BACTERIAL ATP SYNTHASES

TURINA, MARIA PAOLA;REBECCHI, ALBERTO;D'ALESSANDRO, MANUELA;MELANDRI, BRUNO ANDREA
2006

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 (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.
2006
14th European Bioenergetics Conference 2006 Short Reports
41
41
MODULATION OF PROTON PUMPING EFFICIENCY IN BACTERIAL ATP SYNTHASES / P. Turina; A. Rebecchi; M. D'Alessandro; S. Anefors; B.A. Melandri. - In: BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS. - ISSN 0005-2728. - STAMPA. - 1757:5–6, Supplement(2006), pp. 41-41. (Intervento presentato al convegno 14th European Bioenergetic Conference tenutosi a Moscow, RUSSIA nel JUL 22-27, 2006).
P. Turina; A. Rebecchi; M. D'Alessandro; S. Anefors; B.A. Melandri
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/123038
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