ATP Synthesis by the F 0 F 1 ATP Synthase from Thermophilic Bacillus PS3 Reconstituted into Liposomes with Bacteriorhodopsin

The correlation between the rate of ATP synthesis and light‐induced proton flux was investigated in proteoliposomes reconstituted with bacteriorhodopsin and ATP synthase from therinophilic 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–were obtained at saturating light intensities under a steady‐state pH gradient of about pH 1.25. It was detnonstrated that this was the maxmal pH 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 (equilbrium) 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 F 0 F 1 concentration led to a large increase in the rate of ATP synthesis whereas the inagnitude of pH 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 irnpaired bacteriorhodopsin (Asp96→Asn mutaled bactericirliodopsin) further demonnstreted that this increase in the coupling efficiency could not he related to protein‐protein interactions but rather to bacteriorhodopsin donating H + to the ATP synthase. Increasing the amount of negatively chargcd phospholipids in the proteoliposomes also increased the coupling efficiency between bacteriorhodopsin and ATP synthase at a constant transtnenibrane pH grudient. Similar results were obtained with chloroplast ATP synthase. Furthermore, ATP synthase activities induced by dpH/Ψ 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 F 0 F 1 might exist under specific experimental conditions.