Site-directed mutagenesis of the PsaC subunit of photosystem I -: FB is the cluster interacting with soluble ferredoxin

The two [4Fe-4S] clusters F-A and F-B are the terminal electron accepters 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 Asp(9) and GlU(46), which are located one residue upstream of the first cysteine liganding cluster F-B and F-A, 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 S-fold) was observed by flash absorption spectroscopy for the D9N and D9K mutant PSI complexes. In the FA(2) triple mutant (V49I/K52T/R53Q), modification of residues located next to the F-A cluster leads to partial destabilization of the PSI complex. The electron paramagnetic resonance properties of cluster F-A are affected, and a 3-fold decrease of Fd affinity is observed. The introduction of positively charged residues close to the F-B cluster in the FB, 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 with t1/2 of < 1 and approximate to 4.5 ys) for all mutants except FB1, where Fd reduction is almost monophasic with tM < 1 mu s. These data indicate that F-B is the cluster interacting with Fd and therefore the outermost iron-sulfur cluster of PSI.