A psaC deletion mutant of the unicellular cyanobacterium Synechocystis sp, PCC 6803 was utilized to incorporate site-specific amino acid substitutions in the cysteine residues that ligate the F-A and F-B iron-sulfur clusters in Photosystem I (PS I). Cysteines 14 and 51 of PsaC were changed to aspartic acid (C14D(PsaC), C51D(PsaC), C14D/C51D(PsaC)), serine (C14S(PsaC), C51S(PsaC)), and alanine (C14A(PsaC), C51A(PsaC)), and the properties of F-A and F-B were characterized by electron paramagnetic resonance spectroscopy and time-reserved optical spectroscopy, The C14D(PsaC)-PS I and C14S(PsaC)-PS I complexes showed high levels of photoreduction of F-A with g values of 2.045, 1.944, and 1.852 after illumination at 15 K, but there was no evidence of reduced F-B in the g = 2 region, The C51D(PsaC)-PS I and C51S(PsaC)-PS I complexes showed low levels of photoreduction of F-B with g values of 2.067, 1.931, and 1.881 after illumination at 15 K, but there was no evidence of reduced F-A in the g = 2 region, The presence of F-B was inferred in C14D(PsaC)-PS I and C14S(PsaC)-PS I, and the presence of F-A was inferred in C51D(PsaC)-PS I and C51S(PsaC)-PS I by magnetic interaction in the photoaccumulated spectra and by the equal spin concentration of the irreversible P700(+) cation generated by illumination at 77 K, Flash-induced optical absorbance changes at 298 K in the presence of a fast electron donor indicate that two electron accepters function after F-X in the four mutant PS I complexes at room temperature, These data suggest that a mixed-ligand [4Fe-4S] cluster is present in the mutant sites of CI LX-PS I and C5LX-PS I (where X = D or S), but that the proposed spin state of S = 3/2 renders the resonances undetectable in the g 2 region, The C14A(PsaC)-PS I, C51A(PsaC)-PS I and C14D/C51D(PsaC)-PS I complexes show only the photoreduction of F-X, consistent with the absence of PsaC. These results show that only those PsaC proteins that contain two [4Fe-4S] clusters are capable of assembling onto PS I cores in vivo.