Kless H, Vermaas W
Department of Botany, Arizona State University, Tempe 85287-1601, USA.
Biochemistry. 1996 Dec 24;35(51):16458-64. doi: 10.1021/bi962240q.
Tyr161 of the D1 protein (Yz) is a redox component closely associated with the water-oxidizing complex of photosystem II. Yz reduces the primary donor P680+, and Yzox is then rereduced by the manganese cluster that oxidizes water. We aimed to investigate whether water oxidation by P680+ could occur through an alternative pathway in the absence of Tyr161. For this purpose, combinatorial mutagenesis was performed in residues presumed to be in the environment of Tyr161. Full sequence degeneracy was introduced in two regions of the D1 protein: at codons 157, 158, 160, 162, 163, 164, and 165, which are close to Yz by sequence, and at codons 186-191 which are assumed to be close to Yz in the tertiary structure; at position 161, the nucleotide combinations were designed to not give rise to a Tyr codon. The combinatorial DNA mixture was used to transform an obligate photoheterotrophic mutant (Y161W) of the cyanobacterium Synechocystis sp. PCC 6803, in which Trp at position 161 impairs photosynthetic activity. Transformants were selected in which photoautotrophic growth was restored, resulting in 11 viable mutants. In all of these mutants, however, a Tyr codon was found at position 161, introduced either by complex repair processes or as a result of PCR-induced mutations. Additional mutations found in residues neighboring Tyr161 mostly retained photosystem II properties similar to those of wild type. However, in two of these mutants, FVEYPI and FLVYNI, photoautotrophic growth was impaired and the relative variable fluorescence was reduced. Computer simulations of the environment of Yz suggest that the position of Tyr161 varies with respect to some neighboring residues without major functional consequences. We conclude that Tyr161 fulfills a critical role through its chemical nature and positioning and that this function cannot be substituted by another residue at a nearby position.
D1蛋白（Yz）的Tyr161是一种氧化还原成分，与光系统II的水氧化复合物密切相关。Yz还原初级供体P680+，然后氧化态的Yz被氧化水的锰簇重新还原。我们旨在研究在没有Tyr161的情况下，P680+氧化水是否能通过另一条途径发生。为此，对推测位于Tyr161周围环境中的残基进行了组合诱变。在D1蛋白的两个区域引入了完全序列简并性：在序列上靠近Yz的密码子157、158、160、162、163、164和165处，以及在三级结构中假定靠近Yz的密码子186 - 191处；在位置161处，核苷酸组合设计为不会产生Tyr密码子。该组合DNA混合物用于转化蓝藻集胞藻PCC 6803的专性光异养突变体（Y161W），其中161位的色氨酸会损害光合活性。筛选出恢复了光合自养生长的转化体，得到11个存活突变体。然而，在所有这些突变体中，在161位都发现了一个Tyr密码子，这是由复杂的修复过程或PCR诱导的突变导致的。在Tyr161邻近残基中发现的其他突变大多保留了与野生型相似的光系统II特性。然而，在其中两个突变体FVEYPI和FLVYNI中，光合自养生长受到损害，相对可变荧光降低。Yz环境的计算机模拟表明，Tyr161相对于一些邻近残基的位置有所变化，但没有重大功能影响。我们得出结论，Tyr161通过其化学性质和位置发挥关键作用，并且该功能不能被附近位置的另一个残基替代。
