REDUCED TURNOVER OF THE D1 POLYPEPTIDE AND PHOTOACTIVATION OF ELECTRON-TRANSFER IN NOVEL HERBICIDE-RESISTANT MUTANTS OF SYNECHOCYSTIS SP. PCC-6803

Two missense mutants, A263P and S264P, and a deletion mutant des-Ala26 3, Ser264, have been constructed in the D1 protein of the cyanobacteri um Synechocystis sp PCC 6803. All were expected to induce a significan t conformational change in the Q(B)-binding region of photosystem II ( PSII). Although the des-Ala263. Ser264-D1 mutant accumulated some D1 p rotein in the thylakoid membrane it was unable to grow photoautotrophi cally or evolve oxygen. Thermoluminescence and chlorophyll fluorescenc e studies confirmed that this deletion mutant did not show any functio nal PSII activity. In contrast, [S264P]D1 was able to grow photoautotr ophically and give light-saturated rates of oxygen evolution at 60% of the rate of the wild-type control strain, TC31. The A263P missense mu tant was also able to evolve oxygen at 50% of TC31 rates although it d id not readily grow photoautotrophically. Thermoluminescence. flash ox ygen yield and chlorophyll fluorescence measurements indicated that in both missense mutants electron transfer from Q(A) to Q(B) was signifi cantly impaired in dark adapted cells. However, Q(A) to Q(B) electron transfer could be photoactivated in the mutants by background illumina tion. Both the A263P and S264P mutants also showed an increase in resi stance to the s-triazine family of herbicides although this feature di d not hold for the phenolic herbicide, ioxynil. Of particular interest was that the two missense mutants, especially S264P, possessed a slow er rate of turnover of the D1 protein compared with TC31 and in vivo c ontained detectable levels of a 41-kDa adduct consisting of D1 and the alpha subunit of cytochrome b(559). When protein synthesis was blocke d by the addition of lincomycin, D1 degradation was again slower in S2 64P than TC31. The results are discussed in terms of structural change s in the Q(B)-binding region which affect herbicide and plastoquinone binding and perturb the normal regulatory factors that control the deg radation of the D1 protein and its synchronisation with the synthesis of a replacement D1 protein.