IDENTIFICATION OF DOMAINS ON THE EXTRINSIC 33-KDA PROTEIN POSSIBLY INVOLVED IN ELECTROSTATIC INTERACTION WITH PHOTOSYSTEM-II COMPLEX BY MEANS OF CHEMICAL MODIFICATION

The extrinsic 33-kDa protein of photosystem II (PSII) was modified wit h various reagents, and the resulting proteins were checked for the ab ility to rebind to PSII and to reactivate oxygen evolution, While modi fication of more than eight carboxyl groups of aspartyl and glutamyl r esidues with glycine methyl ester did not affect the rebinding and rea ctivating capabilities, modification of amino groups of lysyl residues with either N-succinimidyl propionate or 2,4,6-trinitrobenzene sulfon ic acid or modification of guanidino groups of arginyl residues with 2 ,3-butanedione resulted in a loss of rebinding and reactivating capabi lities of the 33-kDa protein. Moreover, the number of lysyl and arginy l residues susceptible to modification was significantly decreased whe n the protein was bound to PSII as compared with when it was free in s olution, whereas the number of carboxyl groups modified was little aff ected. These results suggested that positive charges are important for the electrostatic interaction between the extrinsic 33-kDa protein an d PSII intrinsic proteins, whereas negative charges on the protein do not contribute to such interaction. By a combination of protease diges tion and mass spectroscopic analysis, the domains of lysyl residues ac cessible to N-succinimidyl propionate or 2,4,6-trinitrobenzene sulfoni c acid modification only when the 33-kDa protein is free in solution w ere determined to be Lys(4), Lys(20), Lys(66)-Lys(76), Lys(101), Lys(1 05), Lys(130), Lys(159), Lys(186), and Lys(230)-Lys(236). These domain s include those previously reported accessible to N-hydroxysuccinimido biotin only in solution (Frankel and Bricker (1995) Biochemistry 34, 7 492-7497), and may be important for the interaction of the 33-kDa prot ein with PSII intrinsic proteins.