RESONANCE RAMAN CHARACTERIZATION OF REACTION CENTERS IN WHICH BACTERIOCHLOROPHYLL REPLACES THE PHOTOACTIVE BACTERIOPHEOPHYTIN

Q(y)-excitation resonance Raman (RR) spectra are reported for two muta nt reactions centers (RCs) from Rhodobacter sphaeroides in which the p hotoactive bacteriopheophytin (BPhL) is replaced by a bacteriochloroph yll (BChl) molecule, designated by beta(L). One mutation, (M)L214H, yi elds the pigment change via introduction of a histidine residue at pos ition M214. The other mutation, (M)L214H/(L)-E104V, removes the putati ve 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 acc essory BChls (BChl(L,M)) in both beta-mutant and wild-type RCs. The sp ectroscopic data reveal the following: (1) The beta(L) cofactor is a f ive-coordinate BChl molecule with a histidine axial ligand. The confor mation of beta(L) and the strength of the Mg-histidine bond are very s imilar to that of BChl(L,M). (2) The beta(L) cofactor is oriented in t he 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 wi th glutamic acid L104 via the C-9-keto group of the macrocycle. The st rength 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 h ydrogen bonding interaction at the Cs-keto site induces secondary cofa ctor-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 simi lar to those of BChl(L). Consequently, the initial charge-separated in termediate in beta-type RCs is best characterized as a thermal/quantum mechanical admixture of P(+)beta(L)(-) and P(+)BChl(L)(-) (P is the p rimary electron donor), as originally proposed by Kirmaier et al. [(19 95) J. Phys. Chem. 99, 8903-8409].