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

Qy-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 (BChlL,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 BChlL,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 C9-keto group of the macrocycle. The strength of this hydrogen bond is identical to that formed between this protein residue and the C9-keto group of BPhL in wild-type. (4) The hydrogen bonding interaction at the C9-keto site induces secondary cofactor-protein interactions which involve the C2a-acetyl and Cb-alkyl substituent groups. Collectively, the vibrational signatures of beta L indicate that its intrinsic physicochemical properties are very similar to those of BChlL. 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+ BChlL-(P is the primary electron donor), as originally proposed by Kirmaier et al. [(1995) J. Phys. Chem. 99, 8903-8909].