Two-Photon Excited Fluorescence Dynamics in Enzyme-Bound NADH: the Heterogeneity of Fluorescence Decay Times and Anisotropic Relaxation

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.