Tichy M, Vermaas W
Department of Plant Biology, Arizona State University, Tempe 85287-1601, USA.
Biochemistry. 1998 Feb 10;37(6):1523-31. doi: 10.1021/bi9723818.
Regions in the large lumenally exposed region (loop E) of CP47 affect properties of the watersplitting system in photosystem II (PS II). To investigate the role of these regions, we developed a method for functional complementation of obligate photoheterotrophic mutants carrying a deletion in one such region. Using an obligate photoheterotrophic mutant that carries a short deletion (delta (D440-P447) in loop E of CP47, completely degenerate sequences of eight codons in length were introduced at the site of the deletion. Transformants that were complemented to photoautotrophic growth were selected, and 20 such mutants were studied. Sequence analysis revealed that, as expected, in each of them CP47 had been restored to its wild-type length. However, none of the amino acid residues in the deleted region were found to be critical for function. A negatively charged residue at position 440 and a positively charged one at position 444 were favored but not required. Photoautotrophic growth of mutants obtained varied from almost normal to significantly impaired. The mutants contained 20-100% of the amount of PS II present in the wild type, with PS II amounts correlating with the initial rates of oxygen evolution. The mutants had a high rate of photoinactivation, and many mutants showed an up to 1000-fold increase in chloride requirement for photoautotrophic growth. These phenotypic effects were a direct consequence of the CP47 mutations and were not caused by altered binding of one of the extrinsic proteins. No particular amino acid residues in positions 440-447 of CP47 were found to be indispensable for photoautotrophic growth, and many amino acid combinations in this region support PS II function. However, the mutagenized region is shown to interact with the oxygen-evolving site of PS II and appears to have a direct role in chloride binding.
CP47大的管腔暴露区域（环E）中的区域会影响光系统II（PS II）中析水系统的特性。为了研究这些区域的作用，我们开发了一种方法，用于对在其中一个此类区域存在缺失的专性光异养突变体进行功能互补。利用一个在CP47的环E中携带短缺失（Δ（D440 - P447））的专性光异养突变体，在缺失位点引入了长度为八个密码子的完全简并序列。选择互补至光合自养生长的转化体，并对20个这样的突变体进行了研究。序列分析表明，正如预期的那样，在每个突变体中CP47都恢复到了其野生型长度。然而，未发现缺失区域中的任何氨基酸残基对功能至关重要。440位的带负电荷残基和444位的带正电荷残基是有利的，但并非必需。获得的突变体的光合自养生长从几乎正常到显著受损不等。这些突变体中PS II的含量为野生型的20% - 100%，PS II的含量与氧气释放的初始速率相关。这些突变体具有较高的光失活速率，并且许多突变体在光合自养生长中对氯离子的需求增加了高达1000倍。这些表型效应是CP47突变的直接后果，并非由外在蛋白之一的结合改变引起。未发现CP47的440 - 447位中的特定氨基酸残基对光合自养生长是不可或缺的，并且该区域中的许多氨基酸组合都支持PS II的功能。然而，诱变区域显示与PS II的析氧位点相互作用，并且似乎在氯离子结合中具有直接作用。
