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.

Anisotropic relaxation in NADH excited states studied by polarization-modulation pump-probe transient spectroscopy.

We present the results of experimental and theoretical studies of fast anisotropic relaxation and rotational diffusion in the first electron excited state of biological coenzyme NADH in water-ethanol solutions. The experiments have been carried out by means of a novel polarization-modulation transient method and fluorescence polarization spectroscopy. For interpretation of the experimental results a model of the anisotropic relaxation in terms of scalar and vector properties of transition dipole moments has been developed based on the Born-Oppenheimer approximation. This model allows for the description of fast isotropic and anisotropic excited state relaxation under excitation of molecules by ultrafast laser pulses in transient absorption and upconversion experiments. The results obtained suggest that the dynamics of anisotropic rovibronic relaxation in NADH under excitation with 100 fs pump laser pulses can be characterised by a single vibrational relaxation time τv lying in the range of 2-15 ps and a single rotation diffusion time τr lying in the range of 100-450 ps, both depending on ethanol concentration. The dependence of the times τv and τr on the solution polarity (static permittivity) and viscosity has been determined and analyzed. Limiting values of the term P2(cos θ) describing the rotation of the transition dipole moment in the course of vibrational relaxation have been determined from experiments as a function of ethanol concentration and analyzed.