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

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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.