Generation of Bacteriochlorophyll Triplets by the Mutant Reaction Center of Cereibacter sphaeroides G(M71)L/A(M260)W

In the photosynthetic membrane of the purple non-sulfur bacterium Cereibacter sphaeroides, light energy is absorbed by the pigments of light-harvesting complexes and transferred to the photoreaction center (RC), where it is converted into the electrochemical energy of separated charges. Molecules of the bacteriochlorophyll (BChl) dimer P, monomeric BChl, bacteriopheophytin, and ubiquinone are involved in the process of photoinduced electron transfer in RCs. At all stages of the photochemical process, triplet states of pigments are produced with a certain probability, which in native RCs are quenched by the carotenoid molecules. In this study, probability of the bacteriochlorophyll triplet states formation in RCs was artificially increased using site-directed mutagenesis. The amino acid substitution Ala-M260 → Trp leads to the removal of ubiquinone molecule from the active electron transfer branch of the RC, while the Gly-M71 → Leu substitution closes the carotenoid binding site. As a result, electron transfer from the primary donor P to ubiquinone is blocked, leading to rapid recombination of the separated charges and formation of the P triplets. In this work, we investigated thermal stability, spectral and photochemical properties of the mutant RC, as well as EPR spectra, and lifetime of P triplets under various conditions. It was shown that in the double mutant RC, deactivation of the triplet state of P is slowed down, and its interaction with oxygen is observed, apparently generating population of the chemically active state of singlet-excited oxygen. The obtained RCs are of interest for biotechnological applications as potential photosensitizers.