Crystal structure of Psb31, a novel extrinsic protein of photosystem II from a marine centric diatom and implications for its binding and function.

Psb31 is a fifth extrinsic protein found in photosystem II (PSII) of a centric diatom, Chaetoceros gracilis . The protein has been shown to bind directly to PSII in the absence of other extrinsic proteins and serves in part as a substitute for PsbO in supporting oxygen evolution. We report here the crystal structure of Psb31 at a resolution of 1.55 Å. The structure of Psb31 was composed of two domains, one major, N-terminal four helical domain and one minor, flexible C-terminal domain. The four helices in the N-terminal domain were arranged in an up-down-up-down fold, which appeared unexpectedly to be similar to the structure of spinach PsbQ, in spite of their low sequence homology. This suggests that the centric diatom PSII contains another PsbQ-type extrinsic protein in addition to the original PsbQ protein found in the organism. On the other hand, the C-terminal domain of Psb31 has a unique structure composed of one loop and one short helix. Based on these structural analysis and chemical cross-linking experiments, residues responsible for the binding of Psb31 to PSII intrinsic proteins were suggested. The results are discussed in relation to the copy number of extrinsic proteins in higher plant PSII.

Binding and functional properties of five extrinsic proteins in oxygen-evolving photosystem II from a marine centric diatom, Chaetoceros gracilis.

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.

Topological analysis of the extrinsic PsbO, PsbP and PsbQ proteins in a green algal PSII complex by cross-linking with a water-soluble carbodiimide.

The close association of the extrinsic PsbO, PsbP and PsbQ proteins with PSII core subunits in oxygen-evolving PSII complexes from a green alga, Chlamydomonas reinhardtii, was examined by cross-linking experiments with a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The green algal PSII complexes treated with EDC were washed with alkaline Tris to remove the non-cross-linked extrinsic proteins, and then applied to Blue-Native-PAGE to prepare PSII core complexes. The extrinsic proteins cross-linked with PSII core complexes were detected by immunoblotting analysis using antibodies against extrinsic proteins and PSII core subunits. The results showed that the PsbO, PsbP and PsbQ proteins directly associated with CP47, the alpha subunit of cytochrome b559 and a small subunit in PSII core complexes, respectively, through electrostatic interactions. In addition, a cross-linked product between the PsbP and PsbQ proteins was found in alkaline Tris extracts of EDC-treated PSII complexes, and its cross-linked site was examined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF-MS) after digestions with trypsin and endoproteinase Asp-N. The results demonstrated that the positively charged amino group of K176 on the PsbP protein electrostatically interacts with the negatively charged carboxyl group of D28 on the PsbQ protein. These binding properties of the extrinsic proteins in the green algal PSII were compared with those in higher plant PSII.