Resonance Raman characterization of reaction centers in which bacteriochlorophyll replaces the photoactive bacteriopheophytin

Q(y)-excitation resonance Raman (RR) spectra are reported for two mutant reactions centers (RCs) from Rhodobacter sphaeroides in which the photoactive bacteriopheophytin (BPhL) is replaced by a bacteriochlorophyll (BChl) molecule, designated by beta(L). One mutation, (M)L214H, yields the pigment change via introduction of a histidine residue at position M214. The other mutation, (M)L214H/(L)-E104V, removes the putative hydrogen bond between beta(L) and the native glutamic acid residue at position L104. The vibrational signatures of the beta(L) cofactors of the mutants are compared with one another and with those of the accessory BChls (BChl(L,M)) in both beta-mutant and wild-type RCs. The spectroscopic data reveal the following: (1) The beta(L) cofactor is a five-coordinate BChl molecule with a histidine axial ligand. The conformation of beta(L) and the strength of the Mg-histidine bond are very similar to that of BChl(L,M). (2) The beta(L) cofactor is oriented in the protein pocket in a manner similar to that of BPhL of wild-type. (3) The beta(L) cofactor of the (M)L214H mutant forms a hydrogen bond with glutamic acid L104 via the C-9-keto group of the macrocycle. The strength of this hydrogen bond is identical to that formed between this protein residue and the C-9-keto group of BPhL in wild-type. (4) The hydrogen bonding interaction at the Cs-keto site induces secondary cofactor-protein interactions which involve the C-2a-acetyl and C-b-alkyl substituent groups. Collectively, the vibrational signatures of beta(L) indicate that its intrinsic physicochemical properties are very similar to those of BChl(L). Consequently, the initial charge-separated intermediate in beta-type RCs is best characterized as a thermal/quantum mechanical admixture of P(+)beta(L)(-) and P(+)BChl(L)(-) (P is the primary electron donor), as originally proposed by Kirmaier et al. [(1995) J. Phys. Chem. 99, 8903-8409].