PSAD IS REQUIRED FOR THE STABLE BINDING OF PSAC TO THE PHOTOSYSTEM-I CORE PROTEIN OF SYNECHOCOCCUS-SP PCC-6301

The psaC gene product from Synechococcus sp. PCC 7002 and the psaD gene product from Nostoc sp. PCC 8009 were synthesized in Escherichia coli and purified to homogeneity. Incubation of the PsaC apoprotein with the Synechococcus sp. PCC 6301 photosystem I core protein in the presence of FeCl3, Na2S, and beta-mercaptoethanol resulted in a time-dependent transition in the flash-induced absorption change from a 1.2-ms, P700+ F(X)-back-reaction to a long-lived, P700+ [F(A)/F(B)]- back-reaction. ESR studies showed that F(B) and F(A) were photoreduced about equally at 19 K, and while the resonances were shifted upfield, they remained as broad as in the free PsaC holoprotein. When the reconstituted complex was purified in a sucrose gradient containing 0.1% Triton X-100, most of the optical absorption transient reverted to that characteristic of the P700+ F(X)-back-reaction. Addition of purified PsaD to the incubation mixture led to a greater extent of recovery of electron flow to F(A)/F(B) for any given concentration of PsaC. ESR studies showed that F(A), rather than F(B), became the preferred electron acceptor at 19 K; moreover, the resonances moved upfield and sharpened to become nearly identical with those of a control photosystem I complex. When the sample was purified in a sucrose gradient containing 0.1% Triton X-100, the long-lived P700+ [F(A)/F(B)]- optical transient remained stable. Analysis by denaturing polyacrylamide gel electrophoresis showed that the PsaC and PsaD proteins had rebound to the photosystem I core. The data indicate that although PsaC can bind loosely, the presence of PsaD leads to a stable, isolatable photosystem I complex which is spectroscopically indistinguishable from the native complex. Since a PsaC1 fusion protein which contains an amino-terminal extension of five amino acids (MEHSM...) does not bind in the absence of PsaD [Zhao, J., et al. (1990) FEBS Lett. 276, 175-1801, the N-terminus of the PsaC protein could provide a site of interaction with the photosystem I core. We propose that the binding of PsaC to the PsaA/PsaB heterodimer is potentiated by insertion of the F(A)/F(B) clusters into PsaC, and stabilized by the presence of PsaD.