Probing the role of Valine 185 of the D1 protein in the Photosystem II oxygen evolution.

In Photosystem II (PSII), the Mn4CaO5-cluster of the active site advances through five sequential oxidation states (S0 to S4) before water is oxidized and O2 is generated. The V185 of the D1 protein has been shown to be an important amino acid in PSII function (Dilbeck et al. Biochemistry 52 (2013) 6824-6833). Here, we have studied its role by making a V185T site-directed mutant in the thermophilic cyanobacterium Thermosynechococcus elongatus. The properties of the V185T-PSII have been compared to those of the WT*3-PSII by using EPR spectroscopy, polarography, thermoluminescence and time-resolved UV-visible absorption spectroscopy. It is shown that the V185 and the chloride binding site very likely interact via the H-bond network linking TyrZ and the halide. The V185 contributes to the stabilization of S2 into the low spin (LS), S = 1/2, configuration. Indeed, in the V185T mutant a high proportion of S2 exhibits a high spin (HS), S = 5/2, configuration. By using bromocresol purple as a dye, a proton release was detected in the S1TyrZ → S2HSTyrZ transition in the V185T mutant in contrast to the WT*3-PSII in which there is no proton release in this transition. Instead, in WT*3-PSII, a proton release kinetically much faster than the S2LSTyrZ → S3TyrZ transition was observed and we propose that it occurs in the S2LSTyrZ → S2HSTyrZ intermediate step before the S2HSTyrZ → S3TyrZ transition occurs. The dramatic slowdown of the S3TyrZ → S0TyrZ transition in the V185T mutant does not originate from a structural modification of the Mn4CaO5 cluster since the spin S = 3 S3 EPR signal is not modified in the mutant. More probably, it is indicative of the strong implication of V185 in the tuning of an efficient relaxation processes of the H-bond network and/or of the protein.