EPR studies have revealed that removal of calcium using citric acid from the site in spinach photosystem II which is coupled to the photosynthetic O2-evolving process produces a structural change in the manganese cluster responsible for water oxidation. If done in the dark, this yields a modified S1' oxidation state which can be photooxidized above 250 K to form a structurally altered S2' state, as seen by formation of a “modified” multiline EPR signal. Compared to the “normal” S2 state, this new S2'-state EPR signal has more lines (at least 25) and 25% narrower 55Mn hyperfine splittings, indicative of disruption of the ligands to manganese. The calcium-depleted S2' oxidation state is greatly stabilized compared to the native S2 oxidation state, as seen by a large increase in the lifetime of the S2' EPR signal. Calcium reconstitution results in the reduction of the oxidized tyrosine residue 161YD+ (Em ~ 0.7–0.8 V, NHE) within the reaction center D1 protein in both the S1' and S2' states, as monitored by its EPR signal intensity. We attribute this to reduction by Mn. Thus a possible structural role which calcium plays is to bring YD+ into redox equilibrium with the Mn cluster. Photooxidation of S2' above 250 K produces a higher S state (S3 or S4) having a new EPR signal at g = 2.004 ± 0.003 and a symmetric line width of 163 ± 3 G, suggestive of oxidation of an organic donor, possibly an amino acid, in magnetic contact with the Mn cluster. This EPR signal forms in a stoichiometry of 1–2 relative to YD+. This state is photoaccumulated, does not evolve O2, and decays in the dark to the stable S2' state. The enhanced stability and apparent lowered redox potential of the S states can be explained if calcium depletion exposes the Mn cluster to an increased solvent activity, resulting in the binding and hydrolysis of additional water ligands (hydroxo and oxo). The possibility that this causes disproportionation of MnIII to MnII + MnIV is considered on the basis of analogy to the hydrolysis-induced disproportionation observed for synthetic dimanganese complexes. A “gatekeeper” role for calcium in limiting access of substrate water to the catalytic Mn cluster is indicated.
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