FUNCTIONAL RECONSTITUTION OF PHOTOSYSTEM-I REACTION-CENTER FROM CYANOBACTERIUM SYNECHOCYSTIS SP PCC6803 INTO LIPOSOMES USING A NEW RECONSTITUTION PROCEDURE

Photosystem I reaction center from the cyanobacterium Synechocystis sp PCC6803 was reconstituted into phosphatidylcholine/phosphatidic acid liposomes. Liposomes prepared by reverse-phase evaporation were treate d with various amounts of different detergents and protein incorporati on was analyzed at each step of the solubilization process. After dete rgent removal the activities of the resulting proteoliposomes were mea sured. The most efficient reconstitution was obtained by insertion of the protein complex into preformed liposomes destabilized by saturatin g amounts of octylglucoside. In the presence of N-methylphenazonium me thosulfate and ascorbic acid, liposomes containing the reaction center catalyzed a light-dependent net H+ uptake as measured by the 9-aminoa cridine fluorescence quenching and the pH meter. An important benefit of the new reconstitution procedure is that it produces a homogeneous population of large-size proteoliposomes with a low ionic permeability and with a majority inwardly directed H+ transport activity. In optim al conditions, a light-induced Delta pH of about 1.8 units could be su stained at 20 degrees C in the presence of valinomycin. In the absence of valinomycin, a ''back-pressure'' effect of an electrical transmemb rane potential decreased both the rate and the extent of the H+ transp ort. The reaction center was also co-reconstituted with F0F1 H+-ATPase s from chloroplasts and from the thermophilic bacterium, PS3. The co-r econstituted system was shown to catalyze a light-dependent phosphoryl ation which could only be measured in the presence of a high concentra tion of PSI (low lipid/PSI ratios) while no Delta pH could be detected .