Dalla Chiesa M, Friso G, Deák Z, Vass I, Barber J, Nixon P J
Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, UK.
Eur J Biochem. 1997 Sep 15;248(3):731-40. doi: 10.1111/j.1432-1033.1997.00731.x.
Two missense mutants, A263P and S264P, and a deletion mutant des-Ala263, Ser264, have been constructed in the D1 protein of the cyanobacterium Synechocystis sp PCC 6803. All were expected to induce a significant conformational change in the QB-binding region of photosystem II (PSII). Although the des-Ala263, Ser264-D1 mutant accumulated some D1 protein in the thylakoid membrane it was unable to grow photoautotrophically or evolve oxygen. Thermoluminescence and chlorophyll fluorescence studies confirmed that this deletion mutant did not show any functional PSII activity. In contrast, [S264P]D1 was able to grow photoautotrophically and give light-saturated rates of oxygen evolution at 60% of the rate of the wild-type control strain, TC31. The A263P missense mutant was also able to evolve oxygen at 50% of TC31 rates although it did not readily grow photoautotrophically. Thermoluminescence, flash oxygen yield and chlorophyll fluorescence measurements indicated that in both missense mutants electron transfer from QA to QB was significantly impaired in dark adapted cells. However, QA to QB electron transfer could be photoactivated in the mutants by background illumination. Both the A263P and S264P mutants also showed an increase in resistance to the s-triazine family of herbicides although this feature did not hold for the phenolic herbicide, ioxynil. Of particular interest was that the two missense mutants, especially S264P, possessed a slower rate of turnover of the D1 protein compared with TC31 and in vivo contained detectable levels of a 41-kDa adduct consisting of D1 and the alpha subunit of cytochrome b559. When protein synthesis was blocked by the addition of lincomycin, D1 degradation was again slower in S264P than TC31. The results are discussed in terms of structural changes in the QB-binding region which affect herbicide and plastoquinone binding and perturb the normal regulatory factors that control the degradation of the D1 protein and its synchronisation with the synthesis of a replacement D1 protein.
在集胞藻6803（Synechocystis sp PCC 6803）的D1蛋白中构建了两个错义突变体A263P和S264P，以及一个缺失突变体des-Ala263, Ser264。预计所有这些突变体都会在光系统II（PSII）的QB结合区域诱导显著的构象变化。尽管des-Ala263, Ser264-D1突变体在类囊体膜中积累了一些D1蛋白，但它无法进行光合自养生长或释放氧气。热发光和叶绿素荧光研究证实，这个缺失突变体没有显示出任何功能性的PSII活性。相比之下，[S264P]D1能够进行光合自养生长，并且在光饱和时的放氧速率为野生型对照菌株TC31的60%。A263P错义突变体虽然不容易进行光合自养生长，但也能够以TC31速率的50%释放氧气。热发光、闪光产氧量和叶绿素荧光测量表明，在两个错义突变体中，暗适应细胞中从QA到QB的电子传递都受到了显著损害。然而，在突变体中，QA到QB的电子传递可以通过背景光照被光激活。A263P和S264P突变体对三嗪类除草剂的抗性也有所增加，不过酚类除草剂碘苯腈并不具备这一特性。特别有趣的是，与TC31相比，这两个错义突变体，尤其是S264P，D1蛋白的周转速度较慢，并且在体内含有可检测水平的由D1和细胞色素b559的α亚基组成的41 kDa加合物。当通过添加林可霉素阻断蛋白质合成时，S264P中D1的降解再次比TC31慢。本文从QB结合区域的结构变化方面对结果进行了讨论，这些结构变化影响除草剂和质体醌的结合，并扰乱了控制D1蛋白降解及其与替代D1蛋白合成同步的正常调节因子。
