ISOLATION AND CHARACTERIZATION OF MONOMERIC AND DIMERIC CP47-REACTIONCENTER PHOTOSYSTEM-II COMPLEXES

Using the detergents n-dodecyl beta-D-maltoside and heptyl thioglycopy ranoside, a subcore complex of photosystem II (PSII) has been isolated that contains the chlorophyll-binding protein, CP47, and the reaction center components, D1, D2, and cytochrome b(559). We have found, by u sing sucrose density centrifugation, that the resulting preparation co nsisted of a mixture of dimeric and monomeric forms of the CP47 reacti on center (RC) complex, having molecular masses of 410 +/- 30 and 200 +/- 28 kDa, respectively, as estimated by size exclusion chromatograph y. The level of the dimer in the preparation is significantly higher t han the monomeric form. Both the monomer and dimer contain the protein s CP47, D1, and D2 and the alpha- and beta-subunits of cytochrome b(55 9). Analyses by mass spectrometry and N-terminal sequencing showed tha t both forms of the CP47-RC complex contain the products of the psbI, psbT(c) (chloroplast gene), and psbW with molecular masses of 4195,5, 3849.6, and 5927.4 Da, respectively. In contrast to the monomeric form , the CP47-RC dimer contained two extra proteins with low molecular we ights, identified as the products of the psbL and psbK genes having mo lecular masses of 4365.5 and 4292.1, respectively, It was also found t hat the dimer contained slightly more molecules of chlorophyll a (21 /- 2.5) than the monomer (18 +/- 1.5), a characteristic also observed in the room temperature absorption spectrum by comparing the ratio of absorption at 416 and 435 nm. Of particular note was the finding that the dimer, but not the monomer, contained plastoquinone-9 (estimated t o be 1.5 +/- 0.3 molecules per RC). The results indicate that the CP47 -RC monomer is derived from the dimeric form of the complex, and there fore the latter is likely to represent an in vivo conformation. The Ps bT(c) as well as the PsbI and PsbW proteins are identified as being in timately associated with the D1 and D2 proteins, and in the case of th e dimer, importance is placed on the PsbL and PsbK proteins in sustain ing plastoquinone binding and maintenance of the dimeric organization. Assuming only one copy of the alpha- and beta-subunits of cytochrome b(559), the monomeric and dimeric forms of the complex mould be expect ed to contain 21 and 23 x 2 transmembrane helices, respectively.