The extrinsic 33-kDa protein of photosystem II (PSII) was modified with various reagents, and the resulting proteins were checked for the ability to rebind to PSII and to reactivate oxygen evolution. While modification of more than eight carboxyl groups of aspartyl and glutamyl residues with glycine methyl ester did not affect the rebinding and reactivating capabilities, modification of amino groups of lysyl residues with either Nsuccinimidyl propionate or 2,4,6-trinitrobenzene sulfonic acid or modification of guanidino groups of arginyl residues with 2,3-butanedione resulted in a loss of rebinding and reactivating capabilities of the 33-kDa protein. Moreover, the number of lysyl and arginyl residues susceptible to modification was significantly decreased when the protein was bound to PSII as compared with when it was free in solution, whereas the number of carboxyl groups modified was little affected. These results suggested that positive charges are important for the electrostatic interaction between the extrinsic 33-kDa protein and PSII intrinsic proteins, whereas negative charges on the protein do not contribute to such interaction. By a combination of protease digestion and mass spectroscopic analysis, the domains of lysyl residues accessible to N-succinimidyl propionate or 2,4,6-trinitrobenzene sulfonic acid modification only when the 33-kDa protein is free in solution were determined to be
The extrinsic 33-kDa protein of photosystem II (PSII) was intramolecularly cross-linked by a zero-length cross-linker, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The resulting cross-linked 33-kDa protein rebound to urea/NaCl-washed PSII membranes, which stabilized the binding of manganese as effectively as the untreated 33-kDa protein. In contrast, the oxygen evolution was not restored by binding of the cross-linked protein, indicating that the binding and manganese-stabilizing capabilities of the 33-kDa protein are retained but its reactivating ability is lost by intramolecular cross-linking of the protein. From measurements of CD spectra at high temperatures, the secondary structure of the intramolecularly cross-linked 33-kDa protein was found to be stabilized against heat treatment at temperatures 20°C higher than that of the untreated 33-kDa protein, suggesting that structural flexibility of the 33-kDa protein was much decreased by the intramolecular cross-linking. The rigid structure is possibly responsible for the loss of the reactivating ability of the 33-kDa protein, which implies that binding of the 33-kDa protein to PSII is accompanied by a conformational change essential for the reactivation of oxygen evolution. Peptide mapping, N-terminal sequencing, and mass spectroscopic analysis of protease-digested products of the intramolecularly cross-linked 33-kDa protein revealed that cross-linkings occurred between the amino group of Lys 48 and the carboxyl group of Glu
246, and between the carboxyl group of Glu 10 and the amino group of Lys
14. These cross-linked amino acid residues are thus closely associated with each other through electrostatic interactions.
PSII1 is a multisubunit pigment-protein complex which catalyzes the light-driven oxidation of water to molecular oxygen and the reduction of plastoquinone to plastoquinol. The minimum unit for PSII capable of oxygen evolution under physiological conditions contains seven major intrinsic proteins of the reaction center peptides D1 and D2, two apoproteins of cytochrome b 559 , psbI gene product, and two chlorophyll-binding peptides CP47 and CP43, and three extrinsic proteins of 33, 23, and 17 kDa which are associated with the lumenal surface of thylakoid membranes (1-3). The extrinsic 23-and 17-kDa proteins play a role in regulating the PSII affinity for calcium and chloride, and can be removed by treatment with 1.0 -2.0 M NaCl (4 -9). In cyanobacterial and red algal PSII, these two extrinsic proteins are absent, but a low-potential cytochrome c 550 and a 12-kDa protein have been found as the alternative extrinsic components (10 -12). The extrinsic 33-kDa protein, on the other hand, is present in all oxygenic photosynthetic organisms from cyanobacteria to higher plants and plays an important role in stabilizing binding and maintaining functional conformation of the manganese cluster which directly catalyzes the H 2 O-splitting reaction (for reviews, see Refs. 13-15). Removal of the 33-kDa protein from PSII membranes by washing with high concentrati...
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