Pigment Content of D1-D2-Cytochrome b559 Reaction Center Preparations after Removal of CP47 Contamination: An Immunological Study

Abstract

Isolated D1 -D2-cytochrome b559 photosystem I1 reaction center preparations with pigment stoichiometry higher than 4 chlorophylls per 2 pheophytins can be contaminated with CP47 proximal antenna complex. Reaction centers prepared by a modification of the Nanba-Satoh procedure and containing about 6 chlorophylls per 2 pheophytins showed immuno-cross-reactivity when probed with a monoclonal antibody raised against the CP47 polypeptide. Furthermore, they could be fractionated successfully by Superose-12 sieve chromatography into two different populations. The first few fractions off the column contained a more definitive 435 nm shoulder corresponding to increased chlorophyll content, and showed strong immuno-cross-reactivity with the CP47 antibody. The peak fractions off the column displayed a less prominent 435 nm shoulder, and did not cross-react with the antibody. Moreover, when a 6-chlorophyll preparation was mixed with Sepharose beads coupled to CP47 antibody, the eluted material corresponded to a preparation of about 4 chlorophylls per 2 pheophytins and did not show any crossreaction with the antibody against CP47. The amount of CP47 protein in the 6-chlorophyll preparation as quantitated using Coomassie Blue staining or from gel blots was sufficient to account for most of the extra 2 chlorophylls. We conclude that D 1 -D2-cytochrome b559 preparations containing more than 4 chlorophylls per 2 pheophytins can be contaminated with small amounts of CP47-D1 -D2-Cyt b559 complex and that native photosystem I1 reaction centers contain 4 core chlorophylls per 2 pheophytins.

Heterogeneity and Pigment Composition of Isolated Photosystem II Reaction Centers

“…Despite the claims of others (Pueyo et al, 1995), our work confirms earlier (Kobayashi et al, 1990;Gounaris et al, a The ratios were calculated from the density of the bands detected in CP47 and D1 (C-terminal) immunoblots (Figure 4). 1990) and more recent (Eijckelhoff & Dekker, 1995) analyses that the pigment content of the reaction center of PSII (D1/ D2/cytochrome b 559 complex) isolated by ion-exchange chromatography using n-dodecyl β-D-maltoside (Chapman et al, 1989(Chapman et al, , 1991 or other methods (Eijckelhoff & Dekker, 1995) has a stoichiometry of 6 Chl a:2 β-Car:2 Pheo a.…”

“…Separation of these populations was achieved by sucrose density centrifugation, which also yielded a third fraction containing CP47. Thus a minor amount of CP47 does exist in this type of preparation as argued by Pueyo et al (1995), but in our preparations contamination by the apoprotein is less than 2% of that found in the CP47-RC preparation and could only account for a maximum of 0.41 Chl a molecule/RC. This also assumes that CP47 still retains 22 chlorophylls (Alfonso et al, 1994).…”

“…In this report we present detailed analyses of the heterogeneity and pigment composition of PSII RC isolated from pea (Pisum satiVum L.) using ion-exchange chromatography by the method of Chapman et al (199). We show that the immunodetected CP47 contamination of these RC could not contribute to the two extra chlorophylls as is claimed by Pueyo et al (1995) and that the preparation consists of a mixture of monomeric and dimeric complexes.…”

“…It was concluded from these results that the RC itself is associated with 4 Chl a/2 Pheo a molecules and that the higher pigment ratio reported from other laboratories is due to a contamination of the preparation. Using immunoaffinity chromatography to purify further the "6 Chl" RC, Pueyo et al (1995) suggested that the source of chlorophyll contamination is CP47, a Chl a-containing protein very closely associated with PSII RC. As a result, the conclusion has been drawn that the pigment stoichiometry of 6 Chl a:2 Pheo a, published by several groups (Kobayashi et al, 1990;Gounaris et al, 1990;Montoya et al, 1991;Eijckelhoff & Dekker, 1995) is incorrect and that this invalidates the interpretation of many studies of PSII photochemistry using this isolated complex.…”

Purification and Spectroscopic Characterization of Photosystem II Reaction Center Complexes Isolated with or without Triton X-100

“…The close resemblance between the absorption properties of the "4" Chl preparations from this group and our "6" Chl preparations is in agreement with this idea. The A 416 /A 435 ratio of the "4" Chl preparation reported by Pueyo et al (1995) is within the error limit identical to that of RC short (∼1.20). In addition, the spectral fine structure of the 4 K absorption spectrum of this preparation (Chang et al, 1994a) with its deep valley between the 671 and 679 nm absorption bands shows a striking resemblance to that of RC DM ( Figure 7).…”