Comparative Study of Spectral and Functional Properties of Wild Type and Double Mutant H(L173)L/I(L177)H Reaction Centers of the Purple Bacterium Cereibacter sphaeroides.

Previously, we found that in the reaction center (RC) of the purple bacterium Cereibacter sphaeroides, formation of heterodimeric primary electron donor (P) caused by the substitution of His-L173 by Leu, was compensated by the second mutation Ile-L177 - His. Significant changes in the spectral properties, pigment composition, and redox potential of P observed in the H(L173)L RC, are restored to the corresponding characteristics of the native RC in the RC H(L173)L/I(L177)H, with the difference that the energy of the long-wavelength Q_Y optical transition of P increases significantly (by ~75 meV). In this work, it was shown using light-induced difference FTIR spectroscopy that the homodimeric structure of P is preserved in the RC with double mutation with partially altered electronic properties: electronic coupling in the radical-cation of the P⁺ dimer is weakened and localization of the positive charge on one of its halves is increased. Results of the study of the triple mutant RC, H(L173)L/I(L177)H/F(M197)H, are consistent with the assumption that the observed changes in the P⁺ electronic structure, as well as considerable blue shift of the Q_Y P absorption band in the RC H(L173)L/I(L177)H, are associated with modification of the spatial position and/or geometry of P. Using femtosecond transient absorption spectroscopy, it was shown that the mutant H(L173)L/I(L177)H RC retains a sequence of reactions P* → P⁺B_A^- → P⁺H_A^- → P⁺Q_A^- with electron transfer rates and the quantum yield of the final state P⁺Q_A^- close to those observed in the wild-type RC (P* is the singlet-excited state of P; B_A, H_A, and Q_A are molecules of bacteriochlorophyll, bacteriopheophytin, and ubiquinone in the active A-branch of cofactors, respectively). The obtained results, together with the previously published data for the RC with symmetrical double mutation H(M202)L/I(M206)H, demonstrate that by introducing additional point amino acid substitutions, photochemical activity of the isolated RC from C. sphaeroides could be maintained at a high level even in the absence of important structural elements - axial histidine ligands of the primary electron donor P.