Effects of ionizable residues on the absorption spectrum and initial electron-transfer kinetics in the photosynthetic reaction center of Rhodobacter sphaeroides.

Effects of ionizable amino acids on spectroscopic properties and electron-transfer kinetics in the photosynthetic reaction center (RC) of Rhodobacter sphaeroides are investigated by site-directed mutations designed to alter the electrostatic environment of the bacteriochlorophyll dimer that serves as the photochemical electron donor (P). Arginine residues at homologous positions in the L and M subunits (L135 and M164) are changed independently: Arg L135 is replaced by Lys, Leu, Glu, and Gln and Arg M164 by Leu and Glu. Asp L155 also is mutated to Asn, Tyr L164 to Phe, and Cys L247 to Lys and Asp. The mutations at L155, L164, and M164 have little effect on the absorption spectrum, whereas those at L135 and L247 shift the long-wavelength absorption band of P to higher energies. Fits to the ground-state absorption and hole-burned spectra indicate that the blue shift and increased width of the absorption band in the L135 mutants are due partly to changes in the distribution of energies for the zero-phonon absorption line and partly to stronger electron-phonon coupling. The initial electron-transfer kinetics are not changed significantly in most of the mutants, but the time constant increases from 3.0 +/- 0.2 in wild-type RCs to 4.7 +/- 0.2 in C(L247)D and 7.0 +/- 0.3 ps in C(L247)K. The effects of the mutations on the solvation free energies of the product of the initial electron-transfer reaction (P(+)) and the charge-transfer states that contribute to the absorption spectrum ( and ) were calculated by using a distance-dependent electrostatic screening factor. The results are qualitatively in accord with the view that electrostatic interactions of the bacteriochlorophylls with ionized residues of the protein are strongly screened and make only minor contributions to the energetics and dynamics of charge separation. However, the slowing of electron transfer in the Cys L247 mutants and the blue shift of the spectrum in some of the Arg L135 and Cys L247 mutants cannot be explained consistently by electrostatic interactions of the mutated residues with P and B(L); we ascribe these effects tentatively to structural changes caused by the mutations.