ATP SYNTHESIS BY THE F0F1 ATP SYNTHASE FROM THERMOPHILIC BACILLUS PS3RECONSTITUTED INTO LIPOSOMES WITH BACTERIORHODOPSIN .2. RELATIONSHIPSBETWEEN PROTON MOTIVE FORCE AND ATP SYNTHESIS

The correlation between the rate of ATP synthesis and light-induced pr oton flux was investigated in proteoliposomes reconstituted with bacte riorhodopsin and ATP synthase from thermophilic Bacillus PS3. By varia tion of the actinic light intensity it was found that ATP synthase act ivity depended in a sigmoidal manner on the amplitude of the transmemb rane light-induced pH gradient. Maximal rates f ATP synthesis (up to 2 00 nmol ATP . min(-1). mg protein(-1) were obtained at saturating ligh t intensities under a steady-state pH gradient of about pH 1.25. It wa s demonstrated that this was the maximal Delta pH attainable al 40 deg rees C in reconstituted proteoliposomes, due to the feedback inhibitio n of bacteriorhodopsin by the proton gradient it generates. In the abs ence of valinomycin, a small but significant transmembrane electrical potential could develop at 40 degrees C, contributing to an increase i n the rate of APT synthesis. The H+/ATP stoichiometry was measured at the static-head (equilibrium) conditions from the ratio of the phospha te potential to the size of the light-induced pH gradient and a value of about four was obtained under the maximal electrochemical proton gr adient. Increasing the amount of bacteriorhodopsin in the proteoliposo mes at a constant F0F1 concentration led to a large increase in the ra te of ATP synthesis whereas the magnitude of Delta pH remained the sam e or, at very high bacteriorhodopsin levels, decreased. Consequently t he H+/ATP stoichiometry was found to increase significantly with incre asing bacteriorhodopsin content. Reconstitutions with mixtures of nati ve and impaired bacteriorhodopsin (Asp96-->Asn mutated bacteriorhodops in) further demonstrated that this increase in the coupling efficiency could not be related to protein-protein interactions but rather to ba cteriorhodopsin donating H+ to the ATP synthase. Increasing the amount of negatively charged phospholipids in the proteoliposomes also incre ased the coupling efficiency between bacteriorhodopsin and ATP synthas e at a constant transmembrane pH gradient. Similar results were obtain ed with chloroplast ATP synthase. Furthermore, ATP synthase activities induced by Delta pH/Delta Psi transitions were independent of bacteri orhodopsin or anionic lipid levels. These observations were interprete d as indicating that, in bacteriorhodopsin/ATP synthase, proteoliposom es, a localized pathway for coupling light-driven Hi transport by bact eriorhodopsin to ATP synthesis by F0F1 might exist under specific expe rimental conditions.