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Abstract

Mutants of plant light-harvesting complex II (LHC-II) were produced by refolding the complex in vitro from bacterially expressed apoprotein and purified pigments by a method which yields native-like LHC-II in a single step. Amino acid residues known from the structure of the complex [Kühlbrandt, W., et al. (1994) Nature 367, 614−621] to bind chlorophyll (Chl) were replaced with nonbinding residues by site-directed mutagenesis. Recombinant monomeric and trimeric pigment−protein complexes were separated by density gradient centrifugation, and their pigment composition was determined. Six out of nine mutants formed trimers with Chl a:Chl b ratios and Chl contents which suggested they were lacking one Chl a or b per polypeptide. In this way, the identities of Chls a1, a2, a3, b5, and b6 were confirmed as Chl a or b, respectively, whereas Chl b3 in the structure was found to be a Chl a. Absorption and fluorescence emission spectra of the mutant lacking Chl a2 indicated a central role for this Chl in energy transfer to the reaction center.