Mutant trimers of light-harvesting complex II exhibit altered pigment content and spectroscopic features

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 nativelike LHC-II in a single step. Amino acid residues known from the structure of the complex [Kuhlbrandt, 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 Chi a or b per polypeptide. In this way, the identities of Chls a1, n2, n3, b5, and b6 were confirmed as Ch1 a or b, respectively, whereas Ch1 b3 in the structure was found to be a Chi rr, Absorption and fluorescence emission spectra of the mutant lacking Chi a2 indicated a central role for this Chi in energy transfer to the reaction center.