Heterogeneity and pigment composition of isolated photosystem II reaction centers

Photosystem II reaction centers (RC) isolated from peas (Pisum sativum L) purified by ionexchange chromatography were shown, by high-performance liquid chromatography (HPLC) size-exclusion analyses, to consist of a mixture of monomers (180 +/- 20 kDa) and dimers (390 +/- 35 kDa). Both fractions were resolved and purified by sucrose density gradient centrifugation and their homogeneity was demonstrated in size-exclusion HPLC elution profiles. Also present in the nonresolved preparation and the monomeric fraction were low levels of CP47 apoprotein (1.8% and 0.9% apoprotein of that found in a CP47-RC preparation). This CP47 contamination could maximally account for 0.41 and 0.22 Chl/RC, respectively, based on 22 chlorophylls being bound to each CP47 protein. The level of CP47 apoprotein was undetectable in the dimeric fractions. Pigment analysis using reverse-phase HPLC confirmed that contamination by chlorophyll bound to the CP47 apoprotein in the nonresolved RC preparation was low and that the ratio of chlorophyll a to pheophytin a remained 6 when the preparation was separated into its monomeric and dimeric components. We conclude, in agreement with earlier work, that the reaction center of PSII, when isolated using mild detergents and ion-exchange chromatography, contains 6 chlorophyll a/2 pheophytin a. We therefore do not concur with the recent published work of Pueyo et al. [(1995) Biochemistry 34, 15214-15218) that this type of preparation contains 4 chlorophyll a/2 pheophytin a and that the remaining 2 chlorophyll a are due to contamination by CP47.