ANALYSIS OF THE INTERACTION OF WATER WITH THE MANGANESE CLUSTER OF PHOTOSYSTEM-II USING ISOTOPICALLY LABELED WATER

The association of water with the Mn of the water oxidizing complex wa s investigated using (H2O)-O-17- and (H2O)-H-2-reconstituted lyophiliz ed photosystem II particles. The pulsed electron paramagnetic resonanc e (EPR) technique of electron spin echo envelope modulation (ESEEM) wa s used to investigate the interaction of the magnetic H-2 and O-17 nuc lei with the paramagnetic S-2 state of the Mn complex and other photos ystem II components. ESEEM offers a much more specific and sensitive d etection of this type of interaction than continuous wave (CW) EPR. Un like earlier reports using CW EPR, these experiments did not detect an y interaction of water with the multiline EPR signal from the S-2 stat e of the Mn complex. No signals indicating specific interaction of eit her H or O with the multiline signal were detected. Signals due to H-2 and O-17 were detected only at the Larmour frequency, indicating nons pecific ''distant ENDOR'' effects. A weak interaction with O-17 was de tected both in S-1, when the Mn is EPR silent, and in S-2, but only on the high-field side of g = 2. This interaction may be with the Rieske iron-sulfur center in the cytochrome b(6)f complex. The results were the same whether the multiline signal was generated by 200 K illuminat ion of dark-frozen samples, or by room temperature illumination in the presence of the inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DC MU). Illumination at room temperature in the presence of an electron a cceptor to allow multiple turnovers of the system with cycling of the S states did not result in the appearance of any new interactions. The se results appear to exclude close (less than 6 Angstrom) binding of w ater to the Mn center giving rise to the multiline signal, and also to exclude mechanisms in which water oxidation involves the breaking and re-formation of the mu-oxo bridges of the Mn complex. They cannot, ho wever, exclude models in which water binding to the manganese complex and direct oxidation by the manganese complex occur in the higher S st ates, or are catalyzed by one bis(mu-oxo) Mn dimer while oxidizing equ ivalents are accumulated in the S-2 state by a second bis(mu-oxo) Mn d imer.