ESEEM Studies of Substrate Water and Small Alcohol Binding to the Oxygen-Evolving Complex of Photosystem II during Functional Turnover^†

We report the first examination of exchangeable proton and MeOH interactions with the Mn catalytic cluster in photosystem II, under functional flash turnover conditions, using ²H ESEEM spectroscopy on the S₂ and S₀ multiline states. Deuterium-labeled water (D₂O) and methyl d₃-labeled methanol (DMeOH) are employed. It was discovered that a hyperfine resolved multiline S₀ signal could be seen in the presence of D₂O, the hyperfine structure of which depended on the presence or absence of methanol (MeOH). In the presence of DMeOH, significant dipolar coupling of the three methyl deuterons to the multiline centers in the S₂ and S₀ states was seen (S₂, 0.65, 0.39(2) MHz; and S₀, 0.60, 0.37(2) MHz). These are consistent with direct binding of the methoxy fragment to Mn. Assuming terminal Mn−OMe ligation, the couplings indicated a spin projection coefficient (ρ) magnitude of ∼2 for the ligating Mn in both the S₂ and S₀ states, with inferred Mn−O distances of ∼1.9−2.0 Å. In the presence of D₂O, four classes of exchangeable deuterons were identified by ESEEM in S₂ and S₀. Three of these classes (1, 2, and 4) exhibited populations and coupling strengths that were essentially constant under various conditions of sample preparation, illumination turnover, and small alcohol addition. Class 3 could be modeled with constant coupling but a highly variable deuteron population (n₃ ∼ 0−10) depending in part on the preparation used. For all classes, the coupling parameters were very similar in S₂ and S₀. The favored interpretation is that the two strongest coupling classes (1 and 2) represent close binding of one water molecule to a single Mn which has an oxidation state of II in S₀ and III in S₂, and ρ ∼ 2 in both cases. This water is not displaced by MeOH, but either the water or MeOH is singly deprotonated upon MeOH binding. Class 4 represents ∼2 water molecules which are not closely bound to Mn (Mn−deuteron distances of ∼3.7−4.7 Å). Class 3 probably represents protein matrix protons within ∼4 Å of the Mn in the cluster, which can be variably exchanged in different preparations.