PsaC-C14D (cysteine 14 replaced by aspartic acid) contains a [3Fe-4S] and a [4Fe-4S] cluster in the FB and FA sites of the free protein [Yu, L., Zhao, J., Lu, W., Bryant, D. A., & Golbeck, J. H. (1993) Biochemistry 32, 8251-8258]. When PsaC-C14D is rebound to a photosystem I (PS I) core, the g-values of 2.043, 1.939, and 1.853 appear similar to FA in a wild-type PS I complex [Zhao, J. D., Li, N., Warren, P. V., Golbeck, J. H., & Bryant, D. A. (1992) Biochemistry 31, 5093-5099]. The reconstituted PsaC-C14D-PS I complex does not contain a [3Fe-4S] cluster; rather, a set of resonances with a rhombic line shape, a gav of approximately 1.97, and broad line widths indicate the presence of a mixed-ligand [4Fe-4S] cluster, termed FB', in the aspartate site. Both FA and FB' become photoreduced at 15 K, and show an interaction spectrum when reduced within the same reaction center. An electrochemical redox study shows that FA and FB' titrate with midpoint potentials near -600 mV at pH 10.0. Single-turnover flash experiments indicate that FA and FB' function as efficient electron acceptors at room temperature, and NADP+ photoreduction rates are about 70% that of a reconstituted PsaC-PS I complex. A population of S = 3/2, [4Fe-4S] clusters was tentatively identified in the free PsaC-C14D protein by characteristic EPR resonances in the g = 5.3 region.(ABSTRACT TRUNCATED AT 250 WORDS)