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Abstract

The Porphyridium cruentum light harvesting complex (LHC) binds Chl a, zeaxanthin and beta-carotene and comprises at least 6 polypeptides of a multigene family. We describe the first in vitro reconstitution of a red algal light-harvesting protein (LHCaR1) with Chl a/carotenoid extracts from P. cruentum. The reconstituted pigment complex (rLHCaR1) is spectrally similar to the native LHC I, with an absorption maximum at 670 nm, a 77 K fluorescence emission peak at 677 nm (ex. 440 nm), and similar circular dichroism spectra. Molar ratios of 4.0 zeaxanthin, 0.3 beta-carotene and 8.2 Chl a per polypeptide for rLHCaR1 are similar to those of the native LHC I complex (3.1 zeaxanthin, 0.5 beta-carotene, 8.5 Chl a). The binding of 8 Chl a molecules per apoprotein is consistent with 8 putative Chl-binding sites in the predicted transmembrane helices of LHCaR1. Two of the putative Chl a binding sites (helix 2) in LHCaR1 were assigned to Chl b in Chl a/b-binding (CAB) LHC II [Kühlbrandt et al. (1994) Nature 367: 614-21]. This suggests either that discrimination for binding of Chl a or Chl b is not very specific at these sites or that specificity of binding sites evolved separately in CAB proteins. LHCaR1 can be reconstituted with varying ratios of carotenoids, consistent with our previous observation that the carotenoid to Chl ratio is substantially higher in P. cruentum grown under high irradiance. Also notable is that zeaxanthin does not act as an accessory light-harvesting pigment, even though it is highly likely that it occupies the position assigned to lutein in the CAB LHCs.

Chlorophyll and carotenoid binding in a simple red algal light-harvesting complex crosses phylogenetic lines

“…68 kDa, relative to molecular weight markers on sucrose gradients. Those with dinophyte pigments were larger and close to apparent trimers, as was also found with pigments from P. cruentum under the same conditions (22).…”

“…Diatom and dinophyte pigments yielded a brownish-green band, and spinach pigments yielded a green band. Superimposition of pigment bands with Coomassie blue-stained bands, and absence of free pigments on nondenaturing PAGE gels (22) demonstrated adherence of pigments to proteins.…”

“…LHCaR1 (10) was produced in E. coli and the inclusion bodies containing the polypeptide product were isolated according to the method of Paulsen et al (20) as described in Grabowski et al (22).…”

“…Spinach pigment extracts were resuspended in 50% diethyl ether and 50% ethanol: Chl b (from Sigma) was dissolved in 100% diethyl ether. Reconstitution of LHCaR1 was according to Schmid et al (21) as modified by Grabowski et al (22). In the reconstitution with diatom pigments [Chl a͞c ratio (mol͞mol) 7.5], 50 g of Chl a was incubated with 200 g of inclusion body protein, whereas the spinach pigment mixture [Chl a͞b ratio (mol͞mol) 2.8] contained 60 g of Chl and 200 g of protein.…”

“…Biosynthetic pathways for most Chls and carotenoids generally follow phylogenetic lines, and the multigene nature of LHCs makes gene deletion in organisms difficult. In vitro binding of pigments to LHC proteins initially proved its feasibility (19) and has been extensively used for examining LHCs of higher plants (13,20,21), and in a recent report this was extended for the first time to the red algal LHCaR1 (22).…”