The dynamics of polarized fluorescence in NADH in alcohol dehydrogenase (ADH) in buffer solution has been studied using the TCSPC spectroscopy. A global fit procedure was used for determination of the fluorescence parameters from experiment. The interpretation of the results obtained was supported by ab initio calculations of the NADH structure. A theoretical model was developed describing the polarized fluorescence decay in ADH−NADH complexes that considered several interaction scenarios. A comparative analysis of the polarization-insensitive fluorescence decay using multiexponential fitting models has been carried out. As shown, the origin of a significant enhancement of the decay time in the ADH−NADH complex can be attributed to the decrease of nonradiative relaxation rates in the nicotinamide ring in the conditions of the apolar binding site environment. The existence of a single decay time in the ADH−NADH complex in comparison with two decay times observed in free NADH was attributed to a single NADH unfolded conformation in the ADH binding site. Comparison of the experimental data with the theoretical model suggested the existence of an anisotropic relaxation time of about 1 ns that is related with the rotation of fluorescence transition dipole moment due to the rearrangement of the excited state NADH nuclear configuration.

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Combined studies on fluorescence quantum yield in coenzymes NADH and FAD in water-methanol, water-ethanol and water-propylene glycol mixtures and on time-resolved fluorescence of the same molecules by means of the TCSPC method under excitation at 450 and 355 nm have been carried out. A significant difference in the behavior of NADH and FAD quantum yields in different alcohol-water mixtures was observed. The dependence of quantum yield in NADH on alcohol concentration was found to be similar in monohydric alcohols methanol and ethanol and differed from that in polyhydric alcohol propylene glycol. In the case of FAD, the behavior of quantum yield was almost independent of the type of alcohol and exhibited a dramatic increase of 5-6 times with alcohol concentration. The experimental results were analysed using a model based on the quantum mechanical theory developed recently by the authors that took into account radiative and nonradiative relaxation pathways. A generalised expression for the fluorescence quantum yield containing contributions from picosecond and nanosecond nonratiative decay processes in molecular excited states was derived and used for clarification of the role of nanosecond and picosecond relaxation channels in NADH and FAD in various water-alcohol mixtures. The analysis of theoretical expression describing fluorescence decay in polyatomic molecules excited by a short laser pulse resulted in a new insight into the nature of the Decay Associated Spectra (DAS) phenomenon. The contribution from the fast picosecond nonradiative decay in NADH to the quantum yield in water-methanol and water-ethanol mixtures was found to be practically independent of the alcohol concentration and hence of the NADH conformation distribution. This result suggests that the picosecond decay in NADH does not likely occur through electron transfer in the stacking configuration of the nicotinamide and adenine moieties but through other mechanisms. At the same time the contribution of the fast picosecond nonradiative decay in NADH to the quantum yield in water-propylene glycol solutions was found to depend on propylene glycol concentration, therefore the sharp nonlinear increase of the measured quantum yield was associated with decrease of both fast picosecond and relatively slow nanosecong quenching nonradiative rates. In FAD, the contribution of the fast picosecond quenching to the quantum yield was found to depend significantly on the alcohol concentration in all alcoholes under study. This finding suggests that the fast quenching gives a profound contribution to the rise of the measured quantum yield with alcohol concentration and supports the established mechanism of the fluorescence quenching in FAD through electron transfer reaction in the π-stacked conformation between isoalloxazine and adenine moieties.