In vitro reconstitution of the recombinant photosystem II light harvesting complex CP24 and its spectroscopic characterization

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.