Role of an extrinsic 33 kilodalton protein of photosystem II in the turnover of the reaction center-binding protein D1 during photoinhibition.

The reaction center-binding protein D1 of photosystem II (PS II) undergoes rapid turnover under light stress conditions. In the present study, we investigated the role of the extrinsic 33 kDa protein (OEC33) in the early stages of D1 turnover. D1 degradation was measured after strong illumination (1000-5000 microE m-2 S-1) of spinach manganese-depleted, PSII-enriched membrane and core samples in the presence and absence of the OEC33 under aerobic conditions at room temperature. PSII samples lacking the OEC33 were prepared by standard biochemical treatments with Tris or CaCl2/NH2OH while samples retaining the OEC33 were prepared with NH2OH or NaCl/NH2OH. The degradation of D1, monitored by SDS/urea-polyacrylamide gel electrophoresis and Western blotting using specific antibodies against D1, proceeds to a greater extent in NH2OH-treated samples than in Tris-treated samples over a 60 min illumination period. Under the same conditions, significantly more aggregation of D1 occurs in the Tris-treated samples than in the NH2OH-treated samples. The lower level of D1 degradation in Tris-treated samples is not due to secondary proteolysis, as judged from the time course for degradation at 25 degrees C or the degradation pattern at 4 degrees C. Similarly, for NaCl/NH2OH-treated samples, D1 degradation is greater and D1 aggregation less than in CaCl2/NH2OH-treated samples. The effect of the presence of the OEC33 on D1 degradation and aggregation is confirmed by reconstitution experiments in which the isolated OEC33 is restored back to Tris-treated samples. During very strong illumination, significant loss of CP43 also occurs in Tris-treated but not in NH2OH-treated samples. Structural analysis of PS II core complexes by Fourier transform infrared (FT-IR) spectroscopy revealed very little change in the protein secondary structure after 10 min illumination of NH2OH-treated samples while a large 10% decrease of alpha-helix content occurs in Tris-treated samples. On the basis of these results, we suggest that either (1) the OEC33 stabilizes the structural integrity of PS II such that it prevents the photodamaged D1 protein from aggregating with nearby polypeptides and thereby facilitating degradation or (2) the OEC33 specifically stabilizes CP43, a putative D1-specific protease, which normally promotes the efficient degradation of D1.