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 protein CP29 have been studied by using site-directed mutagenesis of pigment-binding 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 chromophore-binding sites. Biochemical 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 domain of the protein are selective for Chl-a, whereas the four peripheral sites (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 compared with glutamate. Xanthophyll site L1 is occupied by lutein, whereas site L2 can bind violaxanthin or neoxanthin. The protein is relatively stable when site L2 site is empty, suggesting that xanthophylls can be exchanged during operation of xanthophyll cycle-dependent photoprotection mechanism. Differential 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.
Bassi
R
R
Università di Verona, Biotecnologie Vegetali, Strada Le Grazie, 37134 Verona, Italy. bassi@sci.univr.it
Croce
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R
Cugini
D
D
Sandonà
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D
D000595:Amino Acid Sequence; D001059:Apoproteins / Q000096:biosynthesis / Q000737:chemistry; D001665:Binding Sites; D003001:Cloning, Molecular; D045342:Light-Harvesting Protein Complexes; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D045332:Photosystem II Protein Complex; D010944:Plants / Q000235:genetics / Q000378:metabolism; D011166:Porphyrins / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D001059:Apoproteins; C069660:CP29 light harvesting complex; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D011166:Porphyrins; D011994:Recombinant Proteins