SITE-DIRECTED MUTATIONS AT RESIDUE-251 OF THE PHOTOSYSTEM-II D1 PROTEIN OF CHLAMYDOMONAS THAT RESULT IN A NONPHOTOSYNTHETIC PHENOTYPE AND IMPAIR D1 SYNTHESIS AND ACCUMULATION

In Cyanobacteria and Chlamydomonas reinhardtii, substitution of valine for alanine at position 251 of the photosystem II D1 protein in the l oop between trans membrane helices IV and V confers resistance to herb icides that reduce photosystem II function and increases sensitivity t o photoinhibition. Using site-directed mutagenesis and chloroplast tra nsformation in Chlamydomonas we have examined further the role of resi due 251 in relation to D1 structure, function, and photosynthetic perf ormance. Of the 12 different amino acid substitutions for Ala(251) int roduced at this position, five (Arg, Asp, Gln, Glu, and His) resulted in a nonphotosynthetic phenotype. Transformants with the Arg(251) subs titution synthesize a normal sized 32-kDa D1 protein with greatly redu ced stability. The Gln, Glu, His, and Asp transformants make a 33-34-k Da form of the D1 protein of varying stability as well as an immunolog ically related polypeptide of 24-25 kDa corresponding to the N-termina l portion of D1 that is unstable and appears to be an aborted D1 trans lation product. All mutant forms of the D1 protein are intrinsic to th e thylakoids. In contrast to previous studies in Cyanobacteria showing that residues in the IV-V loop can be mutated or deleted without loss of photosynthetic competence, our results suggest that Ala(251) has a key role in the structure and function of the IV-V loop region.