Resolution and reconstitution of the cyanobacterial photosystem I complex

We had shown previously that addition of urea to a Synechococcus 6301 Photosystem I complex leads to dissociation of the 8.9 kDa, F_A F_B polypeptide, from the P-700- and F_X-containing Photosystem I core protein (Golbeck et al. (1988) FEBS Lett. 240, 9-14). In the presence of chaotropes, the iron-sulfur clusters in the 8.9 kDa, F_A F_B polypeptide are unstable, and degrade to the level of zero-valence sulfur (Parrett et al. (1989) Biochim. Biophys. Acta 973, 324-332). We now report that addition of FeCl₃, Na₂S, and β-mercaptoethanol to a mixture of the low molecular mass polypeptides and the purified Photosystem I core protein results in complete restoration of light-induced charge separation between P-700 and F_A F_B, including (i) the 30 ms room temperature charge recombination between P-700⁺ and [ F_A F_B]^- and (ii) the characteristic light-induced ESR spectrum of F_A and F_B with g values of 2.05, 1.94, 1.92 and 1.89. Analysis by SDS-PAGE shows that the reconstituted 8.9 kDa, F_A F_B polypeptide has rebound to the Photosystem I core protein. The purified Photosystem I core protein was treated with 3 M urea and 5 mM potassium ferricyanide to oxidatively denature F_X to the level of zero-valence sulfur; light-induced charge separation in the apo-F_X core protein results in a 3 μs optical transient due to the relaxation of the P-700 triplet state. Addition of FeCl₃, Na₂S and β-mercaptoethanol results in restoration of light-induced charge separation between P-700 and F_X, including (i) the 1.2 ms room temperature charge recombination between P-700⁺ and F_X ^- and (ii) the characteristic light-induced ESR resonances of F_X with g values of 2.05, 1.86 and 1.78. Addition of FeCl₃, Na₂S and β-mercaptoethanol to a mixture of the F_X and F_A F_B apoproteins results in reconstitution of electron flow from P-700 to F_A F_B, indicating quantitative reinsertion of the F_X as well as the F_A F_B iron-sulfur clusters and quantitative rebinding of the 8.9 kDa polypeptide to the Photosystem I core protein. This reconstitution technique makes possible novel studies of Photosystem I, including chemical or genetic modification of the F_X or F_A F_B apoproteins followed by reinsertion of the iron-sulfur clusters and rebinding of the low molecular mass polypeptides to produce a functional Photosystem I complex.