Relationship between altered structure and photochemistry in mutant reaction centers in which bacteriochlorophyll replaces the photoactive bacteriopheophytin

Q_y-excitation resonance Raman (RR) spectra are reported for two mutant reaction centers (RCs) from Rhodobacter capsulatus in which the photoactive bacteriopheophytin (BPh_L) is replaced by a bacteriochlorophyll (BChl) molecule, designated β. The pigment change in both mutants is induced via introduction of a histidine residue near the photoactive cofactor. In one mutant, L(M212)H, the histidine is positioned over the core of the cofactor and serves as an axial ligand to the Mg⁺² ion. In the other mutant, F(L121)H/F(L97)V, the histidine is positioned over ring V of the cofactor, which is nominally too distant to permit bonding to the Mg⁺² ion. The salient observations are as follows: (1) The β cofactor in F(L121)H/F(L97)V RCs is a five-coordinate BChl molecule. However, there is no evidence for the formation of a Mg-His bond. This bond is either much weaker than in the L(M212)H RCs or completely absent, the latter implying coordination by an alternative ligand. The different axial ligation for β in the F(L121)H/F(L97)V versus L(M212)H RCs in turn leads to different conformations of the BChl macrocycles. (2) The C₉-keto group of β in F(L121)H/F(L97)V RCs is free of hydrogen bonding interactions, unlike the L(M212)H RCs in which the C₉-keto of β is hydrogen bonded to Glu L104. The interactions between other peripheral substituents of β and the protein are also different in the F(L121)H/F(L97)V RCs versus L(M212)H RCs. Accordingly, the position and orientation of β in the protein is different in the two β-containing RCs. Nonetheless, previous studies have shown that the primary electron transfer reactions are very similar in the two mutants but differ in significant respects compared to wild-type RCs. Collectively, these observations indicate that changes in the conformation of a photoactive tetrapyrrole macrocycle or its interactions with the protein do not necessarily lead to significantly perturbed photochemistry and do not underlie the altered primary events in beta-type RCs. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 346–357, 1999