Stabilization of the Mn cluster by Y_D tyrosine in oxygen evolving complex of photosystem II

本文以单细胞绿色衣藻Chlamydiumreinhardtii为材料，研究了光系统Ⅱ中具有氧化还原活性的Y_D酪氨酸残基的功能。利用自行研制的高灵敏度Joliot型O_2电极，通过分析闪光诱导的光合放氧过程，监测了光合放氧过程中S态的转变过程，使我们能够直接测量培养藻细胞的闪光O_2模式，而不需要进一步的冷凝。在突变体细胞中，从O_2到S_1的暗衰变中未检测到快衰变成分，而慢衰变成分与野生型细胞相比正常，表明Y_D酪氨酸直接参与了快衰变过程。闪光O_2模式即使在长时间的黑暗培养后也没有变化，表明Y_D对S_1和S_0之间的氧化还原平衡没有贡献。因此，S_1比S_0具有更高的稳定性主要归因于它们的热力学性质。结果表明，Y_D不影响Mn团簇在黑暗中的稳定性。事实上，通过镍亲和柱层析，从突变体中制备了具有高O_2活性的PS II核心。该制剂可用于光系统II的物理化学研究，包括ESR和FTIR。

Function of a redox active Y_D tyrosine residue in photosystem II has been studied by the use of the unicellular green alga Chlamydomonas reinhardtii, in which the tyrosine for Y_D (the 160th tyrosine of the D2 protein) was replaced by phenylalanine by site-directed mutagenesis using His-tagged psbD plasmid. The processes of S state transition in photosynthetic oxygen evolution was monitored by analyzing the flash-induced oxygen evolution using a home-made high sensitive Joliot-type O_2 electrode, which enable us to measure the flash O_2 pattern of cultured algal cells directly without further condensation. In the mutant cells, no fast component was detected in the dark decay from O_2 to S_1 but slow decaying component was normal as compared with the wild-type cells, indicating the direct involvement of Y_D tyrosine in the fast decaying process. The flash O_2 pattern did not change even after prolonged dark incubation, indicating that Y_D does not contribute the redox equilibration between S_1 and S_0. Therefore, higher stability of S_1 than S_0 is predominantly ascribed to their thermodynamic natures.Furthermore, the absence of Y_D tyrosine did not affect the O_2 capability after long dark incubation. The results show that Y_D is not involved in the stability of The Mn cluster in the dark. In fact, PS II cores with high O_2 activity were prepared from the mutants by means of Ni-affinity column chromatography. This preparation is useful for the physicochemical studies, including ESR and FTIR, on photosystem II.