In Photosystem II (PSII), the Mn4CaO5-cluster of the active site advances through five sequential oxidation states (S-0 to S-4) before water is oxidized and O-2 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 Tyr(z) and the halide. The V185 contributes to the stabilization of S-2 into the low spin (LS), S = 1/2, configuration. Indeed, in the V185T mutant a high proportion of S-2 exhibits a high spin (HS), S = 5/2, configuration. By using bromocresol purple as a dye, a proton release was detected in the S(1)Tyr(Z)center dot -\textgreater S(2)(LS)Tyr(Z) 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 S(2)(LS)Tyr(z)center dot -\textgreater S(3)Tyr(Z) transition was observed and we propose that it occurs in the S(2)(LS)Tyr(Z)center dot -\textgreater S(2)(HS)Tyr(Z). intermediate step before the S(2)(HS)Tyr(Z)center dot -\textgreater S(3)Tyr(Z) transition occurs. The dramatic slowdown of the S(3)Tyr(Z)center dot -\textgreater S(0)Tyr(Z) transition in the V185T mutant does not originate from a structural modification of the Mn4CaO5 cluster since the spin S = 3 S-3 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.