Evidence for an excited-state reaction contributing to NADH fluorescence

Area of Science:

• Biochemistry
• Biophysics
• Spectroscopy

Background:

• Reduced nicotinamide adenine dinucleotide (NADH) is a crucial biological cofactor.
• Understanding NADH fluorescence dynamics is key to studying enzyme kinetics and mechanisms.
• Previous studies indicated simpler fluorescence decay patterns for NADH.

Purpose of the Study:

• To investigate the fluorescence intensity decay of NADH.
• To analyze the fluorescence properties of the ternary complex of NADH with horse liver alcohol dehydrogenase and iso-butyramide.
• To elucidate the underlying mechanisms responsible for observed fluorescence heterogeneity.

Main Methods:

• Fluorescence intensity decay measurements of NADH.
• Excitation and emission wavelength-dependent analysis.
• Kinetic analysis of the ternary complex formation and its fluorescence decay.

Main Results:

• NADH fluorescence decay exhibited greater heterogeneity in both aqueous and organic solvents than previously reported.
• Formation of the ternary complex necessitated a three-exponent fit for the decay data.
• A sign change in the decay-associated spectrum for the shortest lifetime was observed across the emission spectrum, indicating a complex process.

Conclusions:

• The observed fluorescence heterogeneity suggests complex microenvironments or dynamic processes involving NADH.
• The ternary complex formation triggers a reversible excited-state reaction.
• This reaction leads to the generation of at least one fluorescent product, altering NADH's fluorescence signature.