The secondary electron donor, Tyr Z , is implicated in tuning the primary charge separation and the water oxidation in active photosystem II (PSII). Two types of mechanisms have been proposed to explain the function of Tyr Z . One is that Tyr Z tunes the water oxidation through the direct interaction with substrate water molecules; the other is that Tyr Z is located in a hydrophobic environment without interacting with H 2 O, and controls the water oxidation by tuning the strength of the hydrogen bond between Tyr Z and His190. Here, methanol was used as a probe to study the possible relationship between Tyr Z and H 2 O by monitoring the Tyr Z oxidation and Tyr Z • reduction at cryogenic temperatures with electron paramagnetic resonance spectroscopy. The oxidation of Tyr Z and reduction of Tyr Z • in both S 2 and S 0 states at 10 K were accelerated by addition of a small amount of methanol (6%).Theoretical studies indicate that Tyr oxidation becomes more difficult if it interacts directly with the methanol molecule; while the decrease of the polarity of its environment accelerates the oxidation of Tyr. Accordingly, CH 3 OH does not directly interact with Tyr Z in active PSII, and the accelerative effect of methanol is caused by the strength increase of the hydrogen bond between Tyr Z and His190, resulting from the decrease of polarity of their environment after the displacement of H 2 O by CH 3 OH inside PSII. Considering the similarity between methanol and water, the results in this study support the model in which Tyr Z does not interact with H 2 O in active PSII. photosystem II, secondary electron donor Tyr Z , methanol, water, electron paramagnetic resonance Citation: Bao H, Zhang C X, Ren Y N, et al. Methanol as a probe to investigate the relationship between the secondary electron donor Tyr Z and the substrate water molecules in active photosystem II.Photosystem II (PSII) is the unique biological system in nature to catalyze the oxidation of water to molecular oxygen by using sunlight [1,2]. The light excitation on the PSII reaction center induces the primary charge separation to form the high oxidizing species, P 680 +• , which drives the water catalytic oxidation at the Mn-cluster [3,4]. The turnover of the Mn-cluster leading to water oxidation involves five different states (S n , n=0−4) [5,6], in which S 0 is the initial state, S 1 is the dark stable state, and S 2 and S 3 are more oxidized states. D 1 -Tyr161 (Tyr Z ) is the secondary electron donor located between the Mn-cluster and P 680 . It forms a hydrogen bond with D 1 -His190. The oxidation of Tyr Z by P 680 +• is accompanied by the release of one proton to His190, thus forms a neutral radical, Tyr Z • [7]. Tyr Z plays crucial roles in tuning the primary charge separation in the reaction center and the water oxidation at Mn-cluster. Therefore, the properties and mechanisms of Tyr Z have been the topics under extensive studies [7]. However, due to its fast turnover and lack of characteristic electronic spectroscopic signatures in active PSII sampl...