Abstract
The light-harvesting chlorophyll a/b protein CP24, a minor subunit of the photosystem II antenna system, is a major violaxanthin-binding protein involved in the regulation of excited state concentration of chlorophyll a. This subunit is poorly characterized due to the difficulty in isolation and instability during purification procedures. We have used an alternative approach in order to gain information on the properties of this protein; the Lhcb6 cDNA has been overexpressed in bacteria in order to obtain the CP24 apoprotein, which was then reconstituted in vitro with xanthophylls, chlorophyll a, and chlorophyll b, yielding a pigment-protein complex with properties essentially identical to the native protein extracted from maize thylakoids. Although all carotenoids were supplied during refolding, the recombinant holoprotein exhibited high selectivity in xanthophyll binding by coordinating violaxanthin and lutein but not neoxanthin or beta-carotene. Each monomer bound a total of 10 chlorophyll a plus chlorophyll b and two xanthophyll molecules. Moreover, the protein could be refolded in the presence of different chlorophyll a to chlorophyll b ratios for yielding a family of recombinant proteins with different chlorophyll a/b ratios but still binding the same total number of porphyrins. A peculiar feature of CP24 was its refolding capability in the absence of lutein, contrary to the case of other homologous proteins, thus showing higher plasticity in xanthophyll binding. These characteristics of CP24 are discussed with respect to its role in binding zeaxanthin in high light stress conditions. The spectroscopic analysis of a recombinant CP24 complex binding eight chlorophyll b molecules and a single chlorophyll a molecule by Gaussian deconvolution allowed the identification of four subbands peaking at wavelengths of 638, 645, 653, and 659 nm, which have an increased amplitude with respect to the native complex and therefore identify the chlorophyll b absorption in the antenna protein environment. Gaussian subbands at wavelengths 666, 673, 679, and 686 nm are depleted in the high chlorophyll b complex, thus suggesting they derive from chlorophyll a.
Highlights
In higher plants, chloroplasts, chlorophyll, and carotenoid molecules are noncovalently bound to specific transmembrane proteins to form light-harvesting complexes called LHCI1 and LHCII
Gaussian Deconvolution Analysis of Native and Recombinant CP24 —The results reported above show that rCP24 complexes can be obtained in vitro, and their chromophore content can be modulated for obtaining a stable complex with altered Chl a to Chl b ratio
The publication of a near atomic structure of LHCII has been a major event in the study of chlorophyll a/b proteins [34], since this structure gives the first insight into pigment and protein organization in the major LHCII complex and in the homologous proteins that contribute to the organization of the photosystem I (PSI) and PSII antenna complexes [40, 41]
Summary
Chloroplasts, chlorophyll, and carotenoid molecules are noncovalently bound to specific transmembrane proteins to form light-harvesting complexes called LHCI1 and LHCII. These antenna complexes efficiently capture the light and deliver the excitation energy, respectively, to photosystem I (PSI) and II (PSII) reaction centers, where electron transport occurs, yielding a trans-thylakoid pH gradient, ATP synthesis, and NADPϩ reduction. The photosystem II light-harvesting complex has been extensively investigated and shown to be composed of four chlorophyll a/b proteins, the major complex (LHCII) binding about 65% of PSII chlorophyll and three minor complexes (called CP24, CP26, and CP29) that together bind about 15% of total PSII chlorophyll [1] These minor chlorophyll proteins appear to be involved in the dissipation of the chlorophyll excitation energy needed to prevent overexcitation and photoinhibition of PS II (see Ref. 2 for a review). Analysis of a recombinant CP24 complex binding mainly Chl b allowed us to identify four Gaussian subbands peaking at 638, 645, 652, and 659 nm, which are present in the native complex, identifying the principle components of Chl b absorption in the antenna protein environment
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