# Redox‐dependent binding and conformational equilibria govern the fluorescence decay of NAD(P)H in living cells

## TL;DR

This study explains how enzyme binding and redox states affect the fluorescence decay of NAD(P)H in living cells, improving its use as a metabolic reporter.

## Contribution

The study identifies how redox states control NAD(P)H binding configurations, influencing fluorescence decay in live cells.

## Key findings

- Time-resolved anisotropy imaging reveals multiple enzyme binding configurations of NAD(P)H with varied fluorescence lifetimes.
- Redox states of NAD and NADP pools govern the equilibrium of these binding configurations.
- These findings provide a foundation for accurate interpretation of NAD(P)H FLIM in metabolic studies.

## Abstract

When probed in living systems using fluorescence lifetime imaging microscopy (FLIM), the emission from reduced nicotinamide adenine dinucleotide (NADH) and its phosphorylated form NADPH have shown promise as sensitive intrinsic reporters of metabolism. However, an incomplete understanding of the biochemical processes controlling their fluorescence decay makes it difficult to draw unambiguous conclusions. Here, we utilised time‐resolved fluorescence anisotropy imaging to identify multiple enzyme binding configurations associated with lifetimes both longer and shorter than unbound NAD(P)H. FLIM, combined with mathematical and computational modelling, revealed that the redox states of the NAD and NADP pools control the steady‐state equilibrium of binding configurations, which in turn determines the observed fluorescence decay. This knowledge will be foundational to developing the accurate interpretation of NAD(P)H FLIM.

In this work, we reveal how different enzyme binding configurations influence the fluorescence decay of NAD(P)H in live cells using time‐resolved anisotropy imaging and fluorescence lifetime imaging microscopy (FLIM). Mathematical modelling shows that the redox states of the NAD and NADP pools govern these configurations, shaping their fluorescence lifetimes. This insight advances the interpretation of NAD(P)H FLIM as a metabolic reporter in living systems.