New evidence on the chloride requirement for photosynthetic O2 evolution has indicated that Cl− facilitates oxidation of the manganese cluster by the photosystem II (PSII) Tyr-Z+ radical. Illumination above 250 K of spinach PSII centers which are inhibited in O2 evolution by either Cl− depletion or P substitution produces a new EPR signal which has magnetic characteristics similar to one recently discovered in samples inhibited by depletion of Ca2+ only [Boussac et al. (1989) Biochemistry 28, 8984; Sivaraja et al. (1989) Biochemistry 28, 9459]. The physiological roles of Cl− and Ca2+ in water oxidation are thus linked. The characteristics include a nearly isotropic g = 2.00 ± 0.005, a symmetric line shape with line width = 16 ± 2 mT, almost stoichiometric spin concentration relative to Try-D+ = 0.6 ± 0.3 spin/PSII, very rapid spin relaxation at all temperatures measured down to 6 K, and an undetectable change in magnetic susceptibility upon formation (> 1 μB2). The signal appears to originate from a spin doublet (radical) in magnetic dipolar contact with a transition-metal ion, most probably a photooxidized protein residue within 10 Å of the Mn cluster (Mn-proximal radical). It is distinct from the three other protein-bound radical-type electron donors found in the PSII reaction center: Tyr-D+, Tyr-Z+, and C+. This signal photoaccumulates to a stable level under continuous illumination at 270 K and decays only after illumination stops. Illumination below 250 K suppresses both photooxidation of the Mn cluster and formation of the Mn-proximal radical, with parallel formation of the C+ radical (0.9-mT line width) in place of the usual Tyr-Z+ signal. Either Tyr-Z+ or the Mn cluster are candidates for oxidation of the Mn-proximal protein residue above 250 K. Single-turnover laser-flash EPR studies above 250 K show that the new signal appears after two flashes, photoaccumulates in the S3′ state, and is blocked from further turnover. Nearly fully recovery of water oxidation, low-temperature electron transfer (Mn → Tyr-Z+), and loss of the Mn-proximal EPR signal occur upon Cl− reconstitution. These observations support earlier studies suggesting that photooxidation of a species other than Mn may occur during normal photochemistry in the native enzyme. F−-substituted PSII centers exhibit a large increase in magnetic susceptibility for the S1′ → S2′ state transition that is indistinguishable from Cl−-reconstituted samples, indicative of an equivalent decrease in magnetic coupling between the Mn ions of the cluster for both halides. Therefore, the S1 → S2 oxidation step in H− substituted centers cannot occur at a magnetically isolated Mn(III) monomer site remote from the Mn cluster, as had been suggested earlier by others on the basis of formation of an EPR signal at g = 4.1. The large increase in magnetic susceptibility is consistent with simultaneous Mn oxidation and magnetic uncoupling of a tri-or tetranuclear Mn cluster on the S1 → S2 transition [Sivaraja et al. (1989) J. Am. Chem. Soc. 111, 3221].
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