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

NADH and NADPH play key roles in the regulation of metabolism. Their endogenous fluorescence is sensitive to enzyme binding, allowing changes in cellular metabolic state to be determined using fluorescence lifetime imaging micro-scopy (FLIM). However, to fully uncover the underlying biochemistry, the relationships between their fluorescence and bind -ing dynamics require greater understanding. Here we accomplish this through time-and polarization-resolved fluorescence and polarized two-photon absorption measurements. Two lifetimes result from binding of both NADH to lactate dehydroge-nase and NADPH to isocitrate dehydrogenase. The composite fluorescence anisotropy indicates the shorter (1.3-1.6 ns) decay component to be accompanied by local motion of the nicotinamide ring, pointing to attachment solely via the adenine moiety. For the longer lifetime (3.2-4.4 ns), the nicotinamide conformational freedom is found to be fully restricted. As full and partial nicotinamide binding are recognized steps in dehydrogenase catalysis, our results unify photophysical, structural, and functional aspects of NADH and NADPH binding and clarify the biochemical processes that underlie their contrasting intracellular lifetimes.