Vibrational and photochemical consequences of an Asp residue near the photoactive accessory bacteriochlorophyll in the photosynthetic reaction center

Q(y)-excitation resonance Raman (RR) and subpicosecond resolution transient absorption (TA) studies are reported for Rhodobacter capsulatus reaction centers (RCs) containing a mutation at M-polypeptide residue 201 near the photoactive accessory bacteriochlorophyll (BChl(L)). The studies focus on the electronic/vibration perturbations induced by replacing the native Gly with an Asp residue and examine the effects of temperature on these perturbations. The RCs include the G(M201)D single and G(M201)D/L(M212)H double mutants. In the double mutant, a BChl molecule (designated beta) replaces the native photoactive bacteriopheophytin (BPhL). Analysis of the crystal structure coordinates of the RC indicates that the oxygens of the carbonyl group of the Asp at position M201 should be within a few angstroms of the oxygen of the C-9-keto group of BCh(L). RR studies on both the G(M201)D and G(M201)D/L(M212)H RCs at room temperature indicate that replacing Gly at position M201 with Asp significantly perturbs the vibrational characteristics of BChlL, and in a manner most consistent with Asp M201 being deprotonated and negatively charged. The negative charge of the carboxyl group of Asp M201 interacts with the pi-electron system of BChL in a relatively nonspecific fashion, diminishing the contribution of charge-separated resonance forms of the C-9-keto group to the electronic structure of the cofactor. The RR results are consistent with the effects of Asp M201 on the primary photochemistry found in earlier TA studies on G(M201)D/L(M212)H RCs, which indicate that the Asp residue raises the free energy of state P(+)BChl(L)(-) (Heller et al. Science 1995, 269, 930-945). The interactions between the C-9-keto group of BChl(L) and the carboxyl of Asp M201 are different at ambient versus low temperatures, as reflected in both the RR spectra and TA data on G(M201)D and G(M201)D/L(M212)H RCs. In particular, as the temperature is reduced, both the vibrational characteristics of BChl(L) and the primary photochemistry in the two mutants become more like those found in RCs without the G(M201)D mutation.