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

TL;DR

This study investigates the fluorescence decay and anisotropic relaxation of NADH bound to enzymes, revealing how enzyme binding alters NADH's fluorescence properties and conformations through experimental and theoretical analysis.

Contribution

The paper introduces a combined experimental and theoretical approach to analyze NADH fluorescence dynamics in enzyme complexes, highlighting the impact of enzyme binding on fluorescence decay times and anisotropic relaxation.

Findings

Enzyme binding increases NADH fluorescence decay time due to reduced non-radiative relaxation.

Bound NADH exhibits a single nanosecond decay time, unlike free NADH's multiple decay times.

Anisotropic relaxation time of about 1 ns is observed, linked to enzyme interactions.

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 \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.