Low-temperature absorbance, fluorescence, linear and circular dichroism spectra were measured for two site-specific mutants (beta His21 --> Ser and beta Arg29 --> Glu) of the peripheral (B800-850) light-harvesting complex of Rb. sphaeroides in order to obtain information on the possible changes in the binding site of the bacteriochlorophyll a pigments. From the absorbance and fluorescence measurements we conclude that when beta His21, the putative ligand of the BChl 800 pigment, is replaced by serine the pigment is not incorporated in the complex. In addition, this modification induces a 4 nm red-shift of the B850 band. Linear and circular dichroism measurements indicate that the specific orientation of the BChl 850 pigments is retained despite the absence of a pigment in the B800 binding-site. A second mutant, in which the conserved arginine at position 29 on the same polypeptide is replaced by glutamate, was also studied. This replacement causes a significant blue shift as well as a marked broadening of of the B800 band, which indicates that this binding site is more heterogeneous in this mutant. The overall orientation however is not drastically changed and energy transfer from the B800 to B850 still takes place efficiently. We conclude that neither residue is exclusively involved in the binding pocket for BChl 800, but both beta His21 and beta Arg29 are important in determining the BChl 800 binding.
