Unidirectional light-dependent proton translocation was demonstrated in a suspension of reconstituted reaction center (RC) vesicles supplemented with cytochrome c and 2,3-dimethoxy-5-methyl-1,4-benzoquinone (UQ0), a lipid- and water-soluble quinone. Proton translocation was detected only at alkaline pH. The pH dependence can be accounted for by the slow redox reaction between the reduced quinone (UQ0H2) and oxidized cytochrome c. This conclusion is based on (i) the pH dependence of partial reactions of the reconstituted proton translocation cycle, measured either optically or electrometrically and (ii) titration studies with cytochrome c and UQ0. At 250 and 25 microM UQ0 and cytochrome c, respectively, maximal proton translocation was observed at pH 9.6. This pH optimum can be extended to a more acidic pH by increasing the concentration of the soluble redox mediators in the reconstituted cyclic electron transfer chain. At the alkaline side of the pH optimum, proton translocation appears to be limited by electron transfer from the endogenous primary to the secondary quinone within the RCs. The light intensity limits the reconstituted proton pump at the optimal pH. The results are discussed in the context of a reaction scheme for the cyclic redox reactions and the associated proton translocation events.