RESONANCE RAMAN CHARACTERIZATION OF REACTION CENTERS WITH AN ASP RESIDUE NEAR THE PHOTOACTIVE BACTERIOPHEOPHYTIN

Q(y)-excitation resonance Raman (RR) studies are reported for a series of Rhodobacter capsulatus reaction centers (RCs) containing mutations at L-polypeptide residue 121 near the photoactive bacteriopheophytin (BPhL), The studies focus on the electronic/structural perturbations o f BPhL induced by replacing the native Phe with an Asp residue. Earlie r work has shown that the electron-transfer properties of F(L121)D RCs are closely related to those of RCs in which BPhL is replaced by bact eriochlorophyll (BChl) (beta-type RCs) or by pheophytin. In addition t o the F(L121)D single mutant, RR studies were performed on the F(L121) D/E(L104)L double mutant, which additionally removes the hydrogen bond between BPhL and the native Glu L104 residue. The vibrational signatu res of BPhL in the single and double mutants containing Asp L121 are c ompared with one another and with those of BPhL in both wild-type and F(L121)L RCs. The replacement of the aromatic Phe residue with Leu has no discernible effect on the vibrational properties of BPhL, a findin g in concert with the previously reported absence of an effect of the mutation on the electron-transfer characteristics of the RC. In contra st, replacement of Phe with Asp significantly perturbs the vibrational characteristics of BPhL, and in a manner most consistent with Asp L12 1 being deprotonated and negatively charged. The negative charge of th e carboxyl group of Asp L121 interacts with the pi-electron system of BPhL in a relatively nonspecific fashion, diminishing the contribution of charge-separated resonance forms of the C-9-keto group to the elec tronic structure of the cofactor. The presence of a negative charge ne ar BPhL is consistent with the known photochemistry of F(L121)D RCs, w hich indicates that the free energy of P+BPhL- is substantially higher than in wild type RCs.