The two [4Fe-4S] clusters FAand FB are the terminal electron acceptors of photosystem I (PSI) that are bound by the stromal subunit PsaC. Soluble ferredoxin (Fd) binds to PSI via electrostatic interactions and is reduced by the outermost iron-sulfur cluster of PsaC. We have generated six site-directed mutants of the green alga Chlamydomonas reinhardtii in which residues located close to the iron-sulfur clusters of PsaC are changed. The acidic residues Asp9 and Glu46, which are located one residue upstream of the first cysteine liganding cluster FB and FA, respectively, were changed to a neutral or a basic amino acid. Although Fd reduction is not affected by the E46Q and E46K mutations, a slight increase of Fd affinity (from 1.3- to 2-fold) was observed by flash absorption spectroscopy for the D9N and D9K mutant PSI complexes. In the FA2 triple mutant (V49I/K52T/R53Q), modification of residues located next to the FA cluster leads to partial destabilization of the PSI complex. The electron paramagnetic resonance properties of cluster FA are affected, and a 3-fold decrease of Fd affinity is observed. The introduction of positively charged residues close to the FB cluster in the FB1 triple mutant (I12V/T15K/Q16R) results in a 60-fold increase of Fd affinity as measured by flash absorption spectroscopy and a larger amount of PsaC-Fd cross-linking product. The first-order kinetics are similar to wild type kinetics (two phases witht Formula of <1 and ≈4.5 μs) for all mutants except FB1, where Fd reduction is almost monophasic witht Formula < 1 μs. These data indicate that FB is the cluster interacting with Fd and therefore the outermost iron-sulfur cluster of PSI.