The terminal part of the electron pathway within the photosystem I (PSI) complex includes two [4Fe-4S] centers, FA and FB, which are coordinated by the PsaC subunit. To gain new insights into the electron transfer mechanisms through PsaC, we have generated three mutant strains of the alga Chlamydomonas reinhardtii in which two positively charged residues, K₅₂ and R₅₃, near the FA center have been altered in different ways. The mutations K₅₂S/R₅₃D and K₅₂P/R₅₃D lead to a strong destabilization of PSI. The third mutant K₅₂S/R₅₃A accumulates PSI to 30% of wild-type levels and shares the same residues between two of the cysteine ligands of FA as the PsaC homologue in the green sulfur bacterium Chlorobium limicola, in which FB has a higher redox potential than FA [Nitschke, W., Feiler, U., & Rutherford, A. W. (1990) Biochemistry29, 3834−3842]. Low-temperature electron paramagnetic resonance (EPR) studies reveal that, in contrast to wild type, FB is preferentially photoreduced in this mutant, as was also observed for C. limicola. The preferential photoreduction of FB could be due to changes in the redox potential of FA and/or to slight structural modifications of the PsaC subunit. However, room temperature optical measurements show that stable charge separation still occurs and, surprisingly, that electron transfer from PSI to ferredoxin proceeds at normal rates in the mutant. As C. limicola, the K₅₂S/R₅₃A and K₅₂S/R₅₃D C. reinhardtii mutants are photosensitive when grown aerobically, but can grow photoautotrophically under anaerobic conditions.