The terminal electron acceptors FA and FB exist as two [4Fe-4S] clusters located on the 8.9-kDa PsaC protein in photosystem I. We have used site-directed mutagenesis to produce a complementary pair of mutant PsaC proteins in which specific cysteine ligands to the [4Fe-4S] clusters were changed to aspartic acid residues. The mutant proteins, denoted C14D and C51D, were overproduced in Escherichia coli; the iron-sulfur clusters were inserted in vitro; and the reconstituted proteins were rebound to the P700-Fx core of Synechococcus sp. PCC 6301 in the presence of the PsaD protein. In complexes reconstituted with C51D a rhombic ESR spectrum with g-values of 2.063, 1.934, and 1.879 in the reduced state identifies the intact [4Fe-4S] cluster as FB, while an intense axial spectrum with g-values of 2.020 and 1.997 in the oxidized state identifies the altered cluster in the aspartate site as a [3Fe-4S] cluster. The [3Fe-4S] cluster corresponding to FA can be reduced chemically with dithionite and photochemically by illumination at room temperature but is not reduced by illumination at 15 K. With reconstituted C14D a rhombic ESR spectrum with g-values of 2.043, 1.942, and 1.853 in the reduced state identified the unaltered [4Fe-4S] cluster as FA, while a complex spectrum with a gz-value of 2.194 and an asymmetric gx,y set of resonances between 2.092 and 1.999 indicates an altered cluster of unknown identity in the site containing the aspartate ligand. The ESR signals arising from the altered cluster corresponding to FB are not diminished by illumination at either room temperature or 15 K. Similar to the behavior of the control complex at 15 K, only 12% of the Fb cluster is photoreduced in the complex reconstituted with C51D, whereas about 73% of the FA cluster is photoreduced in the complex reconstituted with C14D. On the basis of amino acid sequence similarities between PsaC and ferredoxins of known structure, we propose that the FA cluster is liganded by cysteines 21, 48, 51, and 54 while the FB cluster is liganded by cysteines 11, 14, 17, and 58. The ability to photoreduce FA in the presence of a nonfunctional FB indicates that the latter is not an obligatory intermediate in the pathway of electrons to FA.
All Science Journal Classification (ASJC) codes