Hydropathy and Molar Volume Constraints on Combinatorial Mutants of the
Photosynthetic Reaction Center
Robles S.J., Youvan D.C.
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139-4307
Combinatorial cassette mutagenesis was used to substitute randomly nine amino acid
residues in the vicinity of the active branch monomeric 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.