HYDROPATHY AND MOLAR VOLUME CONSTRAINTS ON COMBINATORIAL MUTANTS OF THE PHOTOSYNTHETIC REACTION-CENTER

Combinatorial cassette mutagenesis was used to substitute randomly nine amino acid residues in the vicinity of the active branch munomeric bacteriochlorophyll in the photosynthetic reaction center of Rhodobacter capsulatus. The bacteriochlorophyll environment was targeted because of the potential role of this cofactor in the initial charge separation event of photosynthesis. Mutants with perturbed binding and which have altered energy levels of this chromophore, would be useful for electron transfer studies. Four sites in the M-subunit D-helix and five sites in the L-subunit cd-helix, including residue L153, the axial histidine ligand to the bacteriochlorophyll, were randomly substituted. The cd and D-helix regions were mutagenized independently of each other and simultaneously, resulting in three libraries of mutants. Digital imaging spectroscopy was used to screen photosynthetically selected mutants for ground state absorption spectra in the visible and near-infrared. The functional mutants of each library have distinct spectroscopic characteristics. One unusual spectral phenotype with an absorbance band at 825 nm occurred only in the nine-site library, with a frequency of about one out of 106 mutants. A mutant with an 825 nm band also has an unusual light-minus-dark difference spectra, which does not show a blue shift of the 800 nm absorption band. The mean molar volume and hydropathy of the substitutions occurring in functional mutants are biased towards the mean values of a random dope to various extents, indicating different stringencies at each site for these physicochemical properties.