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 c enters (RCs) containing a mutation at M-polypeptide residue 201 near the ph otoactive accessory bacteriochlorophyll (BChl(L)). The studies focus on the electronic/vibration perturbations induced by replacing the native Gly wit h an Asp residue and examine the effects of temperature on these perturbati ons. The RCs include the G(M201)D single and G(M201)D/L(M212)H double mutan ts. In the double mutant, a BChl molecule (designated beta) replaces the na tive photoactive bacteriopheophytin (BPhL). Analysis of the crystal structu re coordinates of the RC indicates that the oxygens of the carbonyl group o f the Asp at position M201 should be within a few angstroms of the oxygen o f 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 BCh lL, and in a manner most consistent with Asp M201 being deprotonated and ne gatively charged. The negative charge of the carboxyl group of Asp M201 int eracts with the pi-electron system of BChL in a relatively nonspecific fash ion, diminishing the contribution of charge-separated resonance forms of th e C-9-keto group to the electronic structure of the cofactor. The RR result s 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 a l. 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 lo w 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, b oth the vibrational characteristics of BChl(L) and the primary photochemist ry in the two mutants become more like those found in RCs without the G(M20 1)D mutation.