Abstract
Oxygen-evolving photosystem II (PSII) isolated from a marine centric diatom, Chaetoceros gracilis, contains a novel extrinsic protein (Psb31) in addition to four red algal type extrinsic proteins of PsbO, PsbQ', PsbV, and PsbU. In this study, the five extrinsic proteins were purified from alkaline Tris extracts of the diatom PSII by anion and cation exchange chromatographic columns at different pH values. Reconstitution experiments in various combinations with the purified extrinsic proteins showed that PsbO, PsbQ', and Psb31 rebound directly to PSII in the absence of other extrinsic proteins, indicating that these extrinsic proteins have their own binding sites in PSII intrinsic proteins. On the other hand, PsbV and PsbU scarcely rebound to PSII alone, and their effective bindings required the presence of all of the other extrinsic proteins. Interestingly, PSII reconstituted with Psb31 alone considerably restored the oxygen evolving activity in the absence of PsbO, indicating that Psb31 serves as a substitute in part for PsbO in supporting oxygen evolution. A significant difference found between PSIIs reconstituted with Psb31 and with PsbO is that the oxygen evolving activity of the former is scarcely stimulated by Cl(-) and Ca(2+) ions but that of the latter is largely stimulated by these ions, although rebinding of PsbV and PsbU activated oxygen evolution in the absence of Cl(-) and Ca(2+) ions in both the former and latter PSIIs. Based on these results, we proposed a model for the association of the five extrinsic proteins with intrinsic proteins in diatom PSII and compared it with those in PSIIs from the other organisms.
Highlights
Induced electron transfer from water to plastoquinone, with the concomitant production of molecular oxygen
In green algal photosystem II (PSII), the PsbP and PsbQ proteins are only partially released by 1 M NaCl-wash, and most of the three extrinsic proteins are released by treatments with 2.6 M urea plus 0.2 M NaCl, 1 M Tris, or 1 M CaCl2, a small amount of the PsbO protein remains bound after these treatments (7)
To obtain a higher level of restoration of the oxygen evolution, we screened various conditions for reconstitution, and we found that the oxygen evolution restored to 46 –50% of the original activity when reconstitution experiments were performed in buffer B containing 10 M 2,6-dichloroindophenol, 2 mM MnCl2, 10 mM MgCl2, and 10 mM CaCl2 on ice at room light (7– 8 mol photons/m2/s) (Table 1), which is comparable with the restoration level observed in spinach, green algal, Euglena, and red algal PSIIs
Summary
Preparation of Oxygen-evolving PSII from a Marine Centric Diatom, C. gracilis—A marine centric diatom, C. gracilis, was grown in artificial seawater as described previously (29). The thylakoid membranes were treated with 1% Triton X-100 in buffer A at 1 mg of chlorophyll (Chl)/ml for 5 min at 0 °C in the dark and fractionated by differential centrifugations according to Ref. 29. The purified PSII was concentrated by centrifugation at 40,000 ϫ g for 20 min after addition of 10% PEG 6000, suspended in a medium containing 0.4 M sucrose and 40 mM MES-NaOH (pH 6.5) (buffer B), and stored at Ϫ196 °C. Purification Procedures of Five Extrinsic Proteins—The crude PSII suspended in 10 mM MES-NaOH (pH 6.5) (buffer C) was treated with 1 M Tris-HCl (pH 8.5) at 0.5 mg of Chl/ml for 1 h on ice in the dark to release all of the extrinsic proteins of PsbO, PsbQЈ, PsbV, Psb[31], and PsbU (29, 30, 33). Assay of Oxygen Evolving Activity—Oxygen evolution was measured with a Clark-type electrode at 25 °C in buffer B with 0.4 mM phenyl-p-benzoquinone as the electron acceptor in the absence or presence of 10 mM NaCl or 5 mM CaCl2
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