Calcium depletion modifies the structure of the photosystem IIO2-evolving complex

A 5 min exposure of photosystem 11 to a pH 3 citric acid solution is a simple method for selective removal of Ca2+ from the O-2-evolving complex. The resulting preparation retains the 23 and 17 kDa extrinsic polypeptides, but the activity of this material is only 10-20% of that of an untreated control sample. Biochemical characterization of citrate-treated photosystem 11 reveals that some reaction centers lose the extrinsic proteins during citrate treatment. Furthermore, a comparison of photosystem 11 preparations treated with citrate, or depleted of 23 and 17 kDa extrinsic polypeptides by high-salt treatment, shows that low concentrations of a small reductant, NH2OH, which has little effect on the activity of intact photosystem II, can reduce and inhibit the Mn cluster in both types of preparations. In contrast, a large reductant, hydroquinone, cannot access the majority Of O-2-evolving centers in citrate-treated preparations, while 23 and 17 kDa-depleted material is rapidly inactivated by the reductant. Incubation of the citrate-treated samples in high (similar to60 mM) concentrations of CaCl2 restores 50% of the lost activity; this Ca2+-reconstituted activity is chelator-insensitive, indicating that rebinding of Ca2+ restores the structural integrity of the O-2-evolving complex. A characterization of Ca2+ and Cl- affinities in steady-state activity assays shows that citrate-treated preparations exhibit a Cl- requirement similar to that of polypeptide-depleted photosystem II, while Ca2+ reactivation Of O-2 evolution appears to occur at two structurally distinct sites. One site exhibits a high Ca2+ affinity, similar to that found in polypeptide-depleted samples, but a second, lower-affinity site also exists, with a K-M that is approximately 10 times greater than that of the high-affinity site, which is associated with centers that retain the extrinsic polypeptides. These data indicate that citrate-induced Ca2+ depletion causes release of the 23 and 17 kDa extrinsic polypeptides from some photosystem II reaction centers, and also modifies the structure of the polypeptide-retaining O-2-evolving centers so that the Mn cluster is exposed to small, but not large, reductants. This change may be due to subtle modifications to the structure of the photosystem 11 extrinsic proteins that produces a new pathway between the solvent and the Mn cluster or, alternatively, to the opening of an existing channel in the intrinsic lumenal polypeptide domain, between the solvent and the Mn cluster, that is normally occluded by a bound Ca2+ atom.