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 reversephase evaporation were treated with various amounts of different detergents and protein incorporation was analyzed at each step of the solubilization process. After detergent removal the activities of the resulting proteoliposomes were measured. The most efficient reconstitution was obtained by insertion of the protein complex into preformed liposomes destabilized by saturating amounts of octylglucoside. In the presence of N-methylphenazonium methosulfate and ascorbic acid, liposomes containing the reaction center catalyzed a light-dependent net H+ uptake as measured by the 9-aminoacridine 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 optimal conditions, a light-induced delta pH of about 1.8 units could be sustained at 20 degrees C in the presence of valinomycin. In the absence of valinomycin, a "back-pressure" effect of an electrical transmembrane potential decreased both the rate and the extent of the H+ transport. The reaction center was also co-reconstituted with F0F1 H(+)-ATPases from chloroplasts and from the thermophilic bacterium, PS3. The co-reconstituted system was shown to catalyze a light-dependent phosphorylation which could only be measured in the presence of a high concentration of PSI (low lipid/PSI ratios) while no delta pH could be detected.
Cladera
J
J
Department de Bioquímica i Biologia Molecular, Universitat Autonoma de Barcelona, Bellaterra, Spain.
Rigaud
J L
JL
Bottin
H
H
Duñach
M
M
D000643:Ammonium Chloride / Q000494:pharmacology; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002784:Cholesterol / Q000494:pharmacology; D000458:Cyanobacteria; D008081:Liposomes / Q000378:metabolism*; D008560:Membrane Fluidity; D008958:Models, Molecular; D009550:Nigericin / Q000494:pharmacology; D045322:Photosynthetic Reaction Center Complex Proteins / Q000378:metabolism*; D045331:Photosystem I Protein Complex; D006180:Proton-Translocating ATPases / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014634:Valinomycin / Q000494:pharmacology
D008081:Liposomes; D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; D000643:Ammonium Chloride; D014634:Valinomycin; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D002784:Cholesterol; D006180:Proton-Translocating ATPases; D009550:Nigericin