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 H-2 ESEEM spectroscopy on the S-2 and S-0 multiline states. Deuterium-labeled water ( D2O) and methyl d(3)-labeled methanol ( DMeOH) are employed. It was discovered that a hyperfine resolved multiline S-0 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 S-2 and S-0 states was seen ( S-2, 0.65, 0.39( 2) MHz; and S-0, 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 ( F) magnitude of similar to 2 for the ligating Mn in both the S-2 and S-0 states, with inferred Mn-O distances of similar to 1.9- 2.0 angstrom. In the presence of D2O, four classes of exchangeable deuterons were identified by ESEEM in S-2 and S-0. 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 ( n(3) similar to 0-10) depending in part on the preparation used. For all classes, the coupling parameters were very similar in S-2 and S-0. 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 S-0 and III in S-2, and rho similar to 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 similar to 2 water molecules which are not closely bound to Mn ( Mn-deuteron distances of similar to 3.7- 4.7 angstrom). Class 3 probably represents protein matrix protons within similar to 4 angstrom of the Mn in the cluster, which can be variably exchanged in different preparations.