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

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.