The role of conformation states in the heterogeneity of fluorescence decay times in FAD in water–alcohol mixtures

We present the results of experimental and theoretical studies of excited state dynamics of flavin adenine dinucleotide (FAD) in water–methanol and water–ethanol mixtures as a function of alcohol concentration. The experimental studies have been carried out by recording time-resolved polarized fluorescence in FAD after excitation with short laser pulses using the time-correlated single photon counting method. The results obtained have shown that in aqueous solution fluorescence decay in FAD could be presented as a sum of four exponents with decay times of 20 ps, 210 ps, 2.70 ns, and 3.85 ns. Addition of methanol, or ethanol only insignificantly affected the decay time values, however caused dramatic changes in the contributions of the exponents to the fluorescence decay signal. Molecular dynamics (MD) simulations and QM/MM calculations in water–methanol and water–ethanol mixtures have been carried out and revealed the existence of three distinct conformation groups of FAD: Stack I, Stack III, and Open, which differ by mutual positions of the adenine and isoalloxazine rings and interaction between them. A model has been developed for elucidation of the excited state dynamics in FAD and of the nature of the heterogeneity of the recorded fluorescence decay times. The model classifies several relaxation channels in FAD after excitation by short laser pulses and suggests that the sub-nanosecond decay times of 20 ps and 210 ps both reflect fast fluorescence quenching due to electron transfer reactions in the vicinity of a conical intersection in the Stack III and Stack I conformations of FAD. The Stack I conformation is mostly stabilized by intramolecular forces due to π-stacking interactions between the adenine and isoalloxazine rings and internal hydrogen bonds, while the Stack III conformation is stabilized to a large extent by hydrogen bonds with external water molecules. It was also suggested that the nanosecond decay times of 2.70 and 3.85 ns were governed mostly by relatively weak non-radiative decay channels from the bottom of the lowest excited electronic state either via direct relaxation to the ground state, or via tunneling to a redox-pair excited state through potential barrier. The decay time of 2.70 ns was shown to refer mainly to folded conformations and the decay time of 3.85 ns to open conformations. [Display omitted] •FAD fluorescence in water exhibits four exponential decay times in ps and ns time-domains.•The decay time contributions change