Journal
BIOCHEMISTRY
Volume 45, Issue 23, Pages 7069-7082Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi052146m
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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.
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