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Free keywords:
Binding Sites/ge [Genetics]; Carotenoids/ch [Chemistry]; Carotenoids/ge [Genetics]; *Chlorophyll/ch [Chemistry]; Chlorophyll/ge [Genetics]; Freezing; Mutagenesis, Site-Directed; Peas; *Photosynthetic Reaction Center, Plant/ch [Chemistry]; Photosynthetic Reaction Center, Plant/ge [Genetics]; Protein Folding; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Spinach; Support, Non-U.S. Gov't
Abstract:
The trimeric main light-harvesting complex (LHC-II) is the only antenna complex of higher plants of which a high-resolution 3D structure has been obtained (Kuhlbrandt, W., Wang, D., and Fujiyoshi, Y. (1994) Nature 367, 614-621) and which can be refolded in vitro from its components. Four different recombinant forms of LHC-II, each with a specific chlorophyll (Chl) binding site removed by site-directed mutagenesis, were refolded from heterologously overexpressed apoprotein, purified pigments, and lipid. Absorption spectra of mutant LHC-II were measured in the temperature range from 4 to 300 K and compared to likewise refolded wild-type complex and to native LHC-II isolated from pea chloroplasts. Chls at different binding sites have characteristic, well-defined absorption sub-bands. Mixed occupation of binding sites with Chls a and b is not observed. Temperature-dependent changes of the mutant absorption spectra reveal a consistent shift of the major difference bands but an irregular behavior of minor bands. A model of the spectral substructure of LHC-II is proposed which accounts for the different absorption properties of the 12 individual Chls in the complex, thus establishing a first consistent correlation between the 3D structure of LHC-II and its spectral properties. The spectral substructure is valid for recombinant and native LHC-II, indicating that both have the same spatial arrangement of Chls and that the refolded complex is fully functional.
