Reconstitution of the photosystem I complex from the P700 and F_x-containing reaction center core protein and the F_A/F_B polypeptide

Complete restoration of electron flow from P700 to F_A/F_B was achieved by incubating a P700 and F_x-containing photosystem (PS) I core protein from Synechococcus sp. 6301 with the 8.9 kDa, F_A/F_B polypeptide from spinach. The ESR spectrum of the reconstituted PS I complex shows nearly equal photochemical reduction of F_A and F_B when frozen in darkness and illuminated at 16 K. When illuminated during freezing, both F_A and F_B are quantitatively reduced and the spectrum is nearly indistinguishable from F_A and F_B in the control PS I complex. In the reconstituted PS I complex F_x is photochemically reduced only in the presence of F^-_A and F^-_B, and the high-field resonance appears indistinguishable from F_x in the control PS I complex. Optical flash photolysis after extensive washing confirms the complete restoration of the P700⁺ [F_A/F_BI-back-reaction, indicating quantitative rebinding of the 8.9 kDA polypeptide. This procedure represents the first reconstitution of the PS I complex from a purified PS I core protein and an isolated 8.9 kDa, F_A/F_B polypeptide, and makes possible independent manipulation of the two subunits that carry the entire electron acceptor system of PS I.