Carotenoid binding sites in LHCIIb - Relative affinities towards major xanthophylls of higher plants

The major light-harvesting complex of photosystem II can be reconstituted i n vitro from its bacterially expressed apoprotein with chlorophylls a and b and neoxanthin, violaxanthin, lutein, or zeaxanthin as the only xanthophyl l. Reconstitution of these one-carotenoid complexes requires low-stringency conditions during complex formation and isolation. Neoxanthin complexes (c ontaining 30-50% of the all-trans isomer) disintegrate during electrophores is, exhibit a largely reduced resistance against proteolytic attack; in add ition, energy transfer from Chi b to Chi a is easily disrupted at elevated temperature. Complexes reconstituted in the presence of either zeaxanthin o r lutein contain nearly two xanthophylls per 12 chlorophylls and are more r esistant against trypsin. Lutein-LHCIIb also exhibits an intermediate maint enance of energy transfer at higher temperature. Violaxanthin complexes app roach a xanthophyll/12 chlorophyll ratio of 3, similar to the ratio in reco mbinant LHCIIb containing all xanthophylls. On the other hand, violaxanthin -LHCIIb exhibits a low thermal stability like neoxanthin complexes, but an intermediate accessibility towards trypsin, similar to lutein-LHCIIb and ze axanthin-LHCIIb. Binary competition experiments were performed with two xan thophylls at varying ratios in the reconstitution. Analysis of the xanthoph yll contents in the reconstitution products yield;ed information about rela tive carotenoid affinities of three assumed binding sites. In lutein/neoxan thin competition experiments, two binding sites showed a strong preference (>200-fold) for lutein, whereas the third binding site had a higher affinit y (25-fold) to neoxanthin. Competition between lutein and violaxanthin gave a similar result, although the specificities were lower: two binding sites have a 36-fold preference for lutein and one has a fivefold preference for violaxanthin. The lowest selectivity was between lutein and zeaxanthin: tw o binding sites had a fivefold higher affinity for lutein and one has a thr eefold higher affinity to zeaxanthin.