Spectroscopic and Theoretical Study on Electronically Modified Chromophores in LOV Domains: 8-Bromo- and 8-Trifluoromethyl-Substituted Flavins

Two chemically synthesized flavin derivatives, 8-trifluoromethyl- and 8-bromoriboflavin (8-CF₃RF and 8-BrRF), were photochemically characterized in H₂O and studied spectroscopically after incorporation into the LOV domain of the blue light photoreceptor YtvA from Bacillus subtilis. The spectroscopic studies were paralleled by high-level quantum chemical calculations. In solution, 8-BrRF showed a remarkably high triplet quantum yield (0.97, parent compound riboflavin, RF: 0.6) and a small fluorescence quantum yield (0.07, RF: 0.27). For 8-CF₃RF, the triplet yield was 0.12, and the fluorescence quantum yield was 0.7. The high triplet yield of 8-BrRF is due to the bromine heavy atom effect causing a stronger spin-orbit coupling. Theoretical calculations reveal that the decreased triplet yield of 8-CF₃RF is due to a smaller charge transfer and a less favorable energetic position of T₂, required for intersystem crossing from S₁ to T₁, as an effect of the electron-withdrawing CF₃ group. The reconstitution of the LOV domain with the new flavins resulted in the typical LOV photochemistry, consisting of triplet state formation and covalent binding of the chromophore, followed by a thermal recovery of the parent state, albeit with different kinetics and photophysical properties. Vision in blue: Flavin derivatives, substituted at position 8 (R=bromo-, trifluoromethyl-) were synthesized and incorporated as chromophores into the blue-light-sensing LOV domain of YtvA from Bacillus subtilis. The resultant strong change in electronegativity significantly modifies the electronic and functional properties of the reconstituted LOV domains.