Identification of Domains on the Extrinsic 33-kDa Protein Possibly Involved in Electrostatic Interaction with Photosystem II Complex by Means of Chemical Modification

Abstract

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

A Domain of the Manganese-stabilizing Protein fromSynechococcus elongatus Involved in Functional Binding to Photosystem II

“…This can be explained by assuming that Lys 160 participates in the electrostatic interaction essential for the function of the protein and that Arg acts only imperfectly for Lys 160 due to the difference in the side group. The corresponding lysyl residue (Lys 159 ) on spinach MSP was unable to participate in an intramolecular charge-pair interaction because the residue was accessible to amino group modifiers when the protein was free in solution (20). It is highly likely, therefore, that Lys 160 contributes to binding of MSP to its functional site by electrostatically interacting with a negatively charged residue on an intrinsic protein of the PSII complex.…”

“…The contribution of arginyl residues to binding of spinach MSP has been suggested by chemical modification of the guanidino group of arginyl residues with 2,3-butanedione (20). None of the six arginyl residues present in MSP reacted with the reagent when the protein was bound to PSII membranes, whereas treatment of the protein in solution led to modification of four arginyl residues, which was accompanied by loss of the ability of the protein to bind to PSII and to reactivate oxygen evolution.…”

“…Analysis of EDC-cross-linked products showed that a charge-pair interaction exists between a charged residue in the Asp 1 -Lys 76 sequence of the spinach MSP and an oppositely charged residue in the Phe 364 -Asp 440 sequence of CP47 (13). Chemical modification with specific reagents showed that six conserved lysyl residues distributed over the entire sequence of spinach MSP are accessible to at least one of the reagents when the protein is free in solution but not when the protein is associated with PSII membranes (19,20). This suggests that these lysyl residues are located in domains of the protein that are in contact with the PSII complex and might participate in intermolecular interaction.…”

“…Mutation of Asp 159 to other residues in Synechocystis MSP affected oxygen evolution significantly (25). On the other hand, in another study, chemical modification of carboxyl groups with glycine methyl ester failed to affect the ability of the spinach protein to reactivate oxygen evolution upon reconstitution at a high protein/PSII ratio (20). Sitedirected mutagenesis of completely or partially conserved aspartyl and glutamyl residues on spinach MSP was performed, but none of the mutations had a dramatic effect (26,27).…”

“…Cross-linking of the Lys 159 -Lys 236 domain of spinach MSP to CP47 with a reagent that cross-links lysyl residues within a 1.2-nm radius has been reported (21). Four arginyl residues of the spinach protein may also be involved in the binding to PSII because they were modified with a specific reagent only when the protein was free in solution (20). Involvement of negatively charged amino acid residues on MSP in binding to PSII was also suggested by EPR studies with * This work was performed under the management of the Research Association for Biotechnology as a part of the R&D Project of Basic Technology for Future Industries, supported by the New Energy and Industrial Technology Development Organization.…”