NAD(P)H binding configurations revealed by time-resolved fluorescence and two-photon absorption.

Area of Science:

• Biochemistry
• Biophysics
• Cellular Metabolism

Background:

• Nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) are vital cofactors in metabolic regulation.
• Their intrinsic fluorescence properties are sensitive to enzyme interactions, enabling metabolic state monitoring via fluorescence lifetime imaging microscopy (FLIM).
• A deeper understanding of the photophysical and binding dynamics of NADH and NADPH is needed to fully elucidate their biochemical roles.

Purpose of the Study:

• To investigate the fluorescence and binding dynamics of NADH and NADPH with their respective enzymes.
• To correlate fluorescence lifetimes with specific binding mechanisms and conformational changes.
• To unify photophysical, structural, and functional insights into NADH and NADPH binding.

Main Methods:

• Time- and polarization-resolved fluorescence spectroscopy.
• Polarized two-photon absorption measurements.
• Analysis of fluorescence anisotropy decay components and lifetimes.

Main Results:

• Two distinct fluorescence lifetimes were observed upon binding of NADH to lactate dehydrogenase and NADPH to isocitrate dehydrogenase.
• Shorter lifetime components (1.3-1.6 ns) correlated with partial nicotinamide ring motion, suggesting adenine-moiety attachment.
• Longer lifetime components (3.2-4.4 ns) indicated fully restricted nicotinamide conformational freedom, consistent with full binding.

Conclusions:

• The study differentiates NADH and NADPH binding mechanisms based on fluorescence lifetime and dynamics.
• Results link photophysical properties to specific catalytic steps in dehydrogenase enzymes.
• This work clarifies the biochemical basis for contrasting intracellular lifetimes of NADH and NADPH.