Intermolecular and Intramolecular Interactions of the 33‐kDa Protein in Photosystem II

Abstract

In this work, cross-linking was observed not only to CP47 but also to a small intrinsic subunit. In addition, through the use of a high-resolution SDS-gel system, three intramolecular cross-linked products of the 33-kDa protein were detected. To search for additional cross-linking sites that might not be accessible to the cross-linker in intact photosystem 11, the isolated 33-kDa protein was activated for cross-linking and subsequently bound to CaC1,-washed photosystem 11. In the complementary experiment, CaC1,-washed photosystem I1 was activated, then reconstituted with the 33-kDa protein. The results of the crosslinking reactions demonstrated that all carboxylic acid groups involved in cross-linking were located on the 33-kDa protein and all primary amines were located on intrinsic membrane proteins. No cross-linking other than those observed in cross-linking experiments with intact photosystem I1 were observed. This indicated that the 33-kDa protein is bound to CP47 and a small subunit but not to the photosystem I1 reaction centre. This observation is consistent with the finding that cross-linking was independent of the presence or absence of the manganese cluster. Possible residues on the 33-kDa protein and CP47 involved in cross-linking are suggested.Keywords: photosystem 11; oxygen evolution; cross-linking ; l-ethyl-3-(3-dimethylaminopropyl)carbodiimide ; protein-protein interaction.Three extrinsic polypeptides that are involved in oxygen evolution are located on the lumenal side of photosystem I1 (PSII) from higher plants. These 33-kDa, 23-kDa and 16-kDa proteins have been extensively studied by dissociation and reconstitution experiments (reviewed by Seidler, 1996). They can be removed by treatment with 1 M Tris, pH 8.0, 2.6 M urea, or with alkaline solutions with concomitant loss of the manganese cluster, the catalytic centre of oxygen evolution. NaCl treatment of PSII removes selectively the 23-kDa and 16-kDa polypeptides. However, the function of these proteins can be replaced by 5 mM Caz+ and 30 mM C1-. Treatment of PSI1 with 1 M CaCI, Inoue, 1983, 1984) or 2.6 M urea and 200 mM NaCl Murata, 1983, 1984) releases all three extrinsic proteins without significant loss of manganese. The activity of PSI1 is lowered to 16-40%, even in the presence of optimal concentrations of Ca2+ and C1-, and the manganese cluster becomes extremely sensitive to light, elevated temperature and endogenous reductants. Rebinding of the 33-kDa protein restores oxygen-evolving activity and the stability of the manganese complex.Despite intensive research, there is still some controversy about where the three extrinsic polypeptides are bound. This question is particularly important for the 33-kDa protein because it appears to be the extrinsic protein which is the most important for oxygen evolution. The strongest evidence has been found for binding of the 33-kDa protein to the chlorophyll-a-binding protein CP47. Removal of the 33-kDa protein was necessary to bind a lysine modifier, N-hydroxysuccinimide-activated biotin to the...

MS-based cross-linking analysis reveals the location of the PsbQ protein in cyanobacterial photosystem II

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Psb27, a transiently associated protein, binds to the chlorophyll binding protein CP43 in photosystem II assembly intermediates

“…Cross-linking of proteins by the water-soluble protein cross-linking agent EDC is a widely used tool to study protein-protein interactions and lowresolution structure determination because it is specific for groups that interact by complementary charges that are in van der Waals' contact with each other (12). It was successfully used to determine close proximities of PsbO and CP47 proteins of PSII (13,14), and these results have subsequently been validated by crystallographic studies of PSII (15).…”

The Use of Advanced Mass Spectrometry to Dissect the Life-Cycle of Photosystem II

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