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

The dynamics of polarized fluorescence in reduced nicotinamide adenine dinucleotide (NADH) at 436~nm under two-photon excitation at 720~nm by femtosecond laser pulses in alcohol dehydrogenase (ADH)-containing buffer solution has been studied experimentally and theoretically. A global fit procedure was used for determination of the fluorescence parameters from experimental data. The interpretation of the experimental results obtained was supported by \emph{ab initio} calculations of NADH structure in solutions. A theoretical model was developed for description of the polarized fluorescence decay in enzyme-NADH binary complexes that considered several possible interaction scenarios. We suggest that the origin of a significant enhancement of the nanosecond decay time value in the ADH-bounded NADH compare with the free NADH can be attributed to the significant decrease of non-radiative relaxation probabilities due to decrease of charges separations in the nicotinamide ring in the conditions of an apolar ADH-NADH binding site environment. The existence of a single decay time in the ADH-NADH complex in the nanosecond time-domain in comparison with two decay times observed in free NADH can be attributed to a single NADH unfolded \emph{trans}-like conformation bounded within the ADH site. Comparison of the experimental data obtained and the theory developed suggested the existence of an anisotropic relaxation time of about 1 ns related most likely with interactions between excited NADH and the ADH binding site that resulted in the rearrangement of nuclear distribution and rotation of fluorescence transition dipole moment.