Mutational analysis of a higher plant antenna protein provides identification of chromophores bound into multiple sites

The chromophore-binding properties of the higher plant light-harvesting pro tein CP29 have been studied by using site-directed mutagenesis of pigment-b inding residues. Overexpression of the apoproteins in bacteria was followed by reconstitution in vitro with purified pigments, thus obtaining a family of mutant CP29 proteins lacking individual chromophorebinding sites. Bioch emical characterization allowed identification of the eight porphyrins and two xanthophyll-binding sites. It is shown that the four porphyrin-binding sites (A1, A2, A4, and A5) situated in the central, twofold-symmetrical dom ain of the protein are selective for Chi-a, whereas the Tour peripheral sit es (A3, B3, B5, and B6) have mixed Chl-a-Chl-b specificity, Within a site, porphyrin coordination by glutamine increases affinity for Chl-b as compare d with glutamate, Xanthophyll site LI is occupied by lutein, whereas site L 2 can bind violaxanthin or neoxanthin, The protein is relatively stable whe n site L2 site is empty, suggesting that xanthophylls can be exchanged duri ng operation of xanthophyll cycle-dependent photoprotection mechanism. Diff erential absorption spectroscopy allowed determination of transition energy levels for individual chromophores, thus opening the way to calculation of energy-transfer rates between Chl in higher plant antenna proteins.