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

Abstract

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 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. The main results obtained are as follows. 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 trans-like confor-