ROLE OF PSII-L PROTEIN (PSBL GENE-PRODUCT) ON THE ELECTRON-TRANSFER IN PHOTOSYSTEM-II COMPLEX .1. OVER-PRODUCTION OF WILD-TYPE AND MUTANT VERSIONS OF PSII-L PROTEIN AND RECONSTITUTION INTO THE PSII CORE COMPLEX

To establish a system for over-production of PSII-L protein which is a component of photosystem II (PSII) complex, a plasmid designated as p MAL-psbL was constructed and expressed in Escherichia coli JM109. A fu sion protein of PSII-L and maltose-binding proteins (53 kDa on SDS-PAG E) was accumulated in E. coli cells to a level of 10% of the total pro tein upon isopropyl-beta-D-thiogalactopyranoside (IPTG) induction. The carboxyl-terminal part of 5.0 kDa was cleaved from the fusion protein and purified by an anion exchange column chromatography in the presen ce of detergents. This 5.0 kDa protein was identified as PSII-L by ami no-terminal amino acid sequence analysis and the chromatographic behav ior on an anion exchange gel. A few types of mutant PSII-L were also p repared by the essentially same procedure except for using plasmids wh ich contain given mutations in psbL gene. Plastoquinone-9 (PQ-9) deple ted PSII reaction center core complex consisting of D1, D2, CP47, cyto chrome b-559 (cyt b-559), PSII-I and PSII-W was reconstituted with PQ- 9 and digalactosyldiglyceride (DGDG) together with the wild-type or mu tant PSII-L produced in E. coli or isolated PSII-L from spinach. Signi ficant difference between the wild-type PSII-L proteins from E. coli a nd spinach was not recognized in the effectiveness to recover the phot oinduced electron transfer activity in the resulting complexes. The an alysis of stoichiometry of PQ-9 per reaction center in the PQ-9 recons tituted PS II revealed that two molecules of PQ-9 were reinserted into a reaction center independent of the presence or absence of PSII-L. T hese results suggest that PSII-L recovers the electron transfer activi ty in the reconstituted RC by a mechanism different from the stabiliza tion of PQ-9 in the QA Site of PSII. Ubiquinone-10 (UQ-10), but not pl astoquinone-2 (PQ-2), substituted PQ-9 for recovering the PSII-L suppo rted electron transfer activity in the reconstituted PSII reaction cen ter complexes. The results obtained with the mutant PSII-L proteins re vealed that the carboxyl terminal part rather than amino terminal part of PSII-L is crucial for recovering the electron transfer activity in the reconstituted complexes.