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

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Tatiana Yu. Fufina¹, Alexey A. Zabelin¹, Ravil A. Khatypov¹, Anton M. Khristin¹, Anatoly Ya. Shkuropatov¹, Lyudmila G. Vasilieva^1,a*

¹Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino Scientific Center for Biological Research, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia

^* To whom correspondence should be addressed.

Received: June 7, 2024; Revised: August 1, 2024; Accepted: August 22, 2024

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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.

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KEY WORDS: photosynthetic reaction center, Cereibacter sphaeroides, ligation of bacteriochlorophyll, primary electron donor, redox potential, thermal stability of membrane proteins, FTIR spectroscopy, femtosecond spectroscopy

DOI: 10.1134/S0006297924100109

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