Isolation and characterization of monomeric and dimeric CP47-reaction center photosystem II complexes

Abstract

Using the detergents n-dodecyl β-D-maltoside and heptyl thioglycopyranoside, 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 b559. We have found, by using sucrose density centrifugation, that the resulting preparation consisted of a mixture of dimeric and monomeric forms of the CP47 reaction center (RC) complex, having molecular masses of 410 ± 30 and 200 ± 28 kDa, respectively, as estimated by size exclusion chromatography. The level of the dimer in the preparation is significantly higher than the monomeric form. Both the monomer and dimer contain the proteins CP47, D1, and D2 and the α- and α-subunits of cytochrome b559. Analyses by mass spectrometry and N- terminal sequencing showed that 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 weights, identified as the products of the psbL and psbK genes having molecular masses of 4365.5 and 4292.1, respectively. It was also found that 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 to 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 therefore the latter is likely to represent an in vivo conformation. The PsbT(c) as well as the PsbI and PsbW proteins are identified as being intimately associated with the D1 and D2 proteins, and in the case of the dimer, importance is placed on the PsbL and PsbK proteins in sustaining plastoquinone binding and maintenance of the dimeric organization. Assuming only one copy of the α- and β-subunits of cytochrome b559, the monomeric and dimeric forms of the complex would be expected to contain 21 and 23 x 2 transmembrane helices, respectively.