Chlorophyll Binding to Monomeric Light-harvesting Complex

The chromophore binding properties of the higher plant light-harvesting complex II have been studied by site-directed mutagenesis of pigment-binding residues. Mutant apoproteins were overexpressed in Escherichia coli and then refolded in vitro with purified chro- mophores to yield holoproteins selectively affected in chlorophyll-binding sites. Biochemical and spectro- scopic characterization showed a specific loss of pig- ments and absorption spectral forms for each mutant, thus allowing identification of the chromophores bound to most of the binding sites. On these bases a map for the occupancy of individual sites by chlorophyll a and chlo- rophyll b is proposed. In some cases a single mutation led to the loss of more than one chromophore indicating that four chlorophylls and one xanthophyll could be bound by pigment-pigment interactions. Differential ab- sorption spectroscopy allowed identification of the Qy transition energy level for each chlorophyll within the complex. It is shown that not only site selectivity is largely conserved between light-harvesting complex II and CP29 but also the distribution of absorption forms among different protein domains, suggesting conserva- tion of energy transfer pathways within the protein and outward to neighbor subunits of the photosystem.