ATP synthesis by the F0F1 ATP synthase from thermophilic Bacillus PS3 reconstituted into liposomes with bacteriorhodopsin: 2. Relationships between proton motive force and ATP synthesis

Bruno Pitard, Peter Richard, Mireia Duñach, Jean Louis Rigaud

Research output: Contribution to journalArticleScientificpeer-review

56 Citations (Scopus)

Abstract

The correlation between the rate of ATP synthesis and light-induced proton flux was investigated in proteoliposomes reconstituted with bacteriorhodopsin and ATP synthase from thermophilic Bacillus PS3. By variation of the actinic light intensity it was found that ATP synthase activity depended in a sigmoidal manner on the amplitude of the transmembrane light-induced pH gradient. Maximal rates of ATP synthesis (up to 200 nmol ATP · min-1 · mg protein-1 were obtained at saturating light intensities under a steady-state pH gradient of about pH 1.25. It was demonstrated that this was the maximal ΔpH attainable at 40°C in reconstituted proteoliposomes, due to the feedback inhibition of bacteriorhodopsin by the proton gradient it generates. In the absence of valinomycin, a small but significant transmembrane electrical potential could develop at 40°C, contributing to an increase in the rate of ATP synthesis. The H+/ATP stoichiometry was measured at the static-head (equilibrium) conditions from the ratio of the phosphate potential to the size of the light-induced pH gradient and a value of about four was obtained under the maximal electrochemical proton gradient. Increasing the amount of bacteriorhodopsin in the proteoliposomes at a constant F0F1 concentration led to a large increase in the rate of ATP synthesis whereas the magnitude of ΔpH remained the same or, at very high bacteriorhodopsin levels, decreased. Consequently the H+/ATP stoichiometry was found to increase significantly with increasing bacteriorhodopsin content. Reconstitutions with mixtures of native and impaired bacteriorhodopsin (Asp96→Asn mutated bacteriorhodopsin) further demonstrated that this increase in the coupling efficiency could not be related to protein-protein interactions but rather to bacteriorhodopsin donating H+ to the ATP synthase. Increasing the amount of negatively charged phospholipids in the proteoliposomes also increased the coupling efficiency between bacteriorhodopsin and ATP synthase at a constant transmembrane pH gradient. Similar results were obtained with chloroplast ATP synthase. Furthermore, ATP synthase activities induced by ΔpH/ΔΨ transitions were independent of bacteriorhodopsin or anionic lipid levels. These observations were interpreted as indicating that, in bacteriorhodopsin/ATP synthase, proteoliposomes, a localized pathway for coupling light-driven H+ transport by bacteriorhodopsin to ATP synthesis by F0F1 might exist under specific experimental conditions.

Original languageEnglish
Pages (from-to)779-788
Number of pages10
JournalEuropean Journal of Biochemistry
Volume235
Issue number3
DOIs
Publication statusPublished - Feb 1996
MoE publication typeNot Eligible

Fingerprint

Bacteriorhodopsins
Proton-Motive Force
Bacilli
Liposomes
Bacillus
Protons
Adenosine Triphosphate
Light
Stoichiometry
Chloroplast Proton-Translocating ATPases
Valinomycin
Proteins
Membrane Potentials

Keywords

  • ATP synthesis
  • Bacteriorhodopsin
  • FF ATP synthase
  • H/ATP stoichiometry
  • Proteoliposomes

Cite this

@article{fb046714e25044608aa966a4c860fb3b,
title = "ATP synthesis by the F0F1 ATP synthase from thermophilic Bacillus PS3 reconstituted into liposomes with bacteriorhodopsin: 2. Relationships between proton motive force and ATP synthesis",
abstract = "The correlation between the rate of ATP synthesis and light-induced proton flux was investigated in proteoliposomes reconstituted with bacteriorhodopsin and ATP synthase from thermophilic Bacillus PS3. By variation of the actinic light intensity it was found that ATP synthase activity depended in a sigmoidal manner on the amplitude of the transmembrane light-induced pH gradient. Maximal rates of ATP synthesis (up to 200 nmol ATP · min-1 · mg protein-1 were obtained at saturating light intensities under a steady-state pH gradient of about pH 1.25. It was demonstrated that this was the maximal ΔpH attainable at 40°C in reconstituted proteoliposomes, due to the feedback inhibition of bacteriorhodopsin by the proton gradient it generates. In the absence of valinomycin, a small but significant transmembrane electrical potential could develop at 40°C, contributing to an increase in the rate of ATP synthesis. The H+/ATP stoichiometry was measured at the static-head (equilibrium) conditions from the ratio of the phosphate potential to the size of the light-induced pH gradient and a value of about four was obtained under the maximal electrochemical proton gradient. Increasing the amount of bacteriorhodopsin in the proteoliposomes at a constant F0F1 concentration led to a large increase in the rate of ATP synthesis whereas the magnitude of ΔpH remained the same or, at very high bacteriorhodopsin levels, decreased. Consequently the H+/ATP stoichiometry was found to increase significantly with increasing bacteriorhodopsin content. Reconstitutions with mixtures of native and impaired bacteriorhodopsin (Asp96→Asn mutated bacteriorhodopsin) further demonstrated that this increase in the coupling efficiency could not be related to protein-protein interactions but rather to bacteriorhodopsin donating H+ to the ATP synthase. Increasing the amount of negatively charged phospholipids in the proteoliposomes also increased the coupling efficiency between bacteriorhodopsin and ATP synthase at a constant transmembrane pH gradient. Similar results were obtained with chloroplast ATP synthase. Furthermore, ATP synthase activities induced by ΔpH/ΔΨ transitions were independent of bacteriorhodopsin or anionic lipid levels. These observations were interpreted as indicating that, in bacteriorhodopsin/ATP synthase, proteoliposomes, a localized pathway for coupling light-driven H+ transport by bacteriorhodopsin to ATP synthesis by F0F1 might exist under specific experimental conditions.",
keywords = "ATP synthesis, Bacteriorhodopsin, FF ATP synthase, H/ATP stoichiometry, Proteoliposomes",
author = "Bruno Pitard and Peter Richard and Mireia Du{\~n}ach and Rigaud, {Jean Louis}",
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ATP synthesis by the F0F1 ATP synthase from thermophilic Bacillus PS3 reconstituted into liposomes with bacteriorhodopsin : 2. Relationships between proton motive force and ATP synthesis. / Pitard, Bruno; Richard, Peter; Duñach, Mireia; Rigaud, Jean Louis.

In: European Journal of Biochemistry, Vol. 235, No. 3, 02.1996, p. 779-788.

Research output: Contribution to journalArticleScientificpeer-review

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T1 - ATP synthesis by the F0F1 ATP synthase from thermophilic Bacillus PS3 reconstituted into liposomes with bacteriorhodopsin

T2 - 2. Relationships between proton motive force and ATP synthesis

AU - Pitard, Bruno

AU - Richard, Peter

AU - Duñach, Mireia

AU - Rigaud, Jean Louis

PY - 1996/2

Y1 - 1996/2

N2 - The correlation between the rate of ATP synthesis and light-induced proton flux was investigated in proteoliposomes reconstituted with bacteriorhodopsin and ATP synthase from thermophilic Bacillus PS3. By variation of the actinic light intensity it was found that ATP synthase activity depended in a sigmoidal manner on the amplitude of the transmembrane light-induced pH gradient. Maximal rates of ATP synthesis (up to 200 nmol ATP · min-1 · mg protein-1 were obtained at saturating light intensities under a steady-state pH gradient of about pH 1.25. It was demonstrated that this was the maximal ΔpH attainable at 40°C in reconstituted proteoliposomes, due to the feedback inhibition of bacteriorhodopsin by the proton gradient it generates. In the absence of valinomycin, a small but significant transmembrane electrical potential could develop at 40°C, contributing to an increase in the rate of ATP synthesis. The H+/ATP stoichiometry was measured at the static-head (equilibrium) conditions from the ratio of the phosphate potential to the size of the light-induced pH gradient and a value of about four was obtained under the maximal electrochemical proton gradient. Increasing the amount of bacteriorhodopsin in the proteoliposomes at a constant F0F1 concentration led to a large increase in the rate of ATP synthesis whereas the magnitude of ΔpH remained the same or, at very high bacteriorhodopsin levels, decreased. Consequently the H+/ATP stoichiometry was found to increase significantly with increasing bacteriorhodopsin content. Reconstitutions with mixtures of native and impaired bacteriorhodopsin (Asp96→Asn mutated bacteriorhodopsin) further demonstrated that this increase in the coupling efficiency could not be related to protein-protein interactions but rather to bacteriorhodopsin donating H+ to the ATP synthase. Increasing the amount of negatively charged phospholipids in the proteoliposomes also increased the coupling efficiency between bacteriorhodopsin and ATP synthase at a constant transmembrane pH gradient. Similar results were obtained with chloroplast ATP synthase. Furthermore, ATP synthase activities induced by ΔpH/ΔΨ transitions were independent of bacteriorhodopsin or anionic lipid levels. These observations were interpreted as indicating that, in bacteriorhodopsin/ATP synthase, proteoliposomes, a localized pathway for coupling light-driven H+ transport by bacteriorhodopsin to ATP synthesis by F0F1 might exist under specific experimental conditions.

AB - The correlation between the rate of ATP synthesis and light-induced proton flux was investigated in proteoliposomes reconstituted with bacteriorhodopsin and ATP synthase from thermophilic Bacillus PS3. By variation of the actinic light intensity it was found that ATP synthase activity depended in a sigmoidal manner on the amplitude of the transmembrane light-induced pH gradient. Maximal rates of ATP synthesis (up to 200 nmol ATP · min-1 · mg protein-1 were obtained at saturating light intensities under a steady-state pH gradient of about pH 1.25. It was demonstrated that this was the maximal ΔpH attainable at 40°C in reconstituted proteoliposomes, due to the feedback inhibition of bacteriorhodopsin by the proton gradient it generates. In the absence of valinomycin, a small but significant transmembrane electrical potential could develop at 40°C, contributing to an increase in the rate of ATP synthesis. The H+/ATP stoichiometry was measured at the static-head (equilibrium) conditions from the ratio of the phosphate potential to the size of the light-induced pH gradient and a value of about four was obtained under the maximal electrochemical proton gradient. Increasing the amount of bacteriorhodopsin in the proteoliposomes at a constant F0F1 concentration led to a large increase in the rate of ATP synthesis whereas the magnitude of ΔpH remained the same or, at very high bacteriorhodopsin levels, decreased. Consequently the H+/ATP stoichiometry was found to increase significantly with increasing bacteriorhodopsin content. Reconstitutions with mixtures of native and impaired bacteriorhodopsin (Asp96→Asn mutated bacteriorhodopsin) further demonstrated that this increase in the coupling efficiency could not be related to protein-protein interactions but rather to bacteriorhodopsin donating H+ to the ATP synthase. Increasing the amount of negatively charged phospholipids in the proteoliposomes also increased the coupling efficiency between bacteriorhodopsin and ATP synthase at a constant transmembrane pH gradient. Similar results were obtained with chloroplast ATP synthase. Furthermore, ATP synthase activities induced by ΔpH/ΔΨ transitions were independent of bacteriorhodopsin or anionic lipid levels. These observations were interpreted as indicating that, in bacteriorhodopsin/ATP synthase, proteoliposomes, a localized pathway for coupling light-driven H+ transport by bacteriorhodopsin to ATP synthesis by F0F1 might exist under specific experimental conditions.

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KW - Bacteriorhodopsin

KW - FF ATP synthase

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