SITE-DIRECTED MUTAGENESIS OF THE PHOTOSYSTEM-I REACTION-CENTER IN CHLOROPLASTS - THE PROLINE-CYSTEINE MOTIF

Site-directed mutagenesis has been used to introduce specific amino ac id changes into the photosystem I reaction center in the green alga Ch lamydomonas reinhardtii. Plasmids containing mutated copies of the chl oroplast psaB gene, encoding a polypeptide of the photosystem I reacti on center heterodimer, were introduced into the chloroplast genome by particle bombardment. Successful transformants were selected by two pr ocedures. The first involved complementation of a nonphotosynthetic mu tant of Chlamydomonas, CC-2341 (ac-u-g-2.3), which has a frameshift mu tation in the psaB gene, and selection of photosynthetic transformants on minimal medium. The second procedure utilized a co-transformation procedure with a plasmid containing a rRNA gene that confers spectinom ycin resistance. Homologous replacement of the psaB gene was confirmed by screening for a unique restriction enzyme site within the transfor ming psaB sequences. These procedures have been used to specifically m utate a highly conserved proline-cysteine motif suggested to be import ant in coordinating the [4Fe-4S] iron-sulfur center F(x). Our results show that the cysteine is essential for assembly of the photosystem I reaction center although the adjacent proline fulfills no identifiable function. The approach described in this paper will be of value to fu ture studies of the structure, function, and assembly of photosystem I .