Chlorophyll binding to monomeric light-harvesting complex. A mutation analysis of chromophore-binding residues.
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 chromophores to yield holoproteins selectively affected in chlorophyll-binding sites. Biochemical and spectroscopic characterization showed a specific loss of pigments 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 chlorophyll 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 absorption spectroscopy allowed identification of the Q(y) 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 conservation of energy transfer pathways within the protein and outward to neighbor subunits of the photosystem.
Remelli
R
R
Università di Verona, Biotecnologie Vegetali, Strada Le Grazie, 37134, Verona, Italy.
Varotto
C
C
Sandonà
D
D
Croce
R
R
Bassi
R
R
D000595:Amino Acid Sequence; D002734:Chlorophyll / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010860:Pigments, Biological / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D045322:Photosynthetic Reaction Center Complex Proteins; D010860:Pigments, Biological; D011994:Recombinant Proteins; D002734:Chlorophyll