NAD(P)H binding configurations revealed by time-resolved fluorescence and two-photon absorption.
Journal
Biophysical journal
ISSN: 1542-0086
Titre abrégé: Biophys J
Pays: United States
ID NLM: 0370626
Informations de publication
Date de publication:
04 04 2023
04 04 2023
Historique:
received:
13
04
2022
revised:
07
09
2022
accepted:
10
02
2023
pmc-release:
04
04
2024
medline:
7
4
2023
pubmed:
17
2
2023
entrez:
16
2
2023
Statut:
ppublish
Résumé
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 microscopy (FLIM). However, to fully uncover the underlying biochemistry, the relationships between their fluorescence and binding 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 dehydrogenase 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.
Identifiants
pubmed: 36793214
pii: S0006-3495(23)00106-6
doi: 10.1016/j.bpj.2023.02.014
pmc: PMC10111271
pii:
doi:
Substances chimiques
NAD
0U46U6E8UK
NADP
53-59-8
Niacinamide
25X51I8RD4
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1240-1253Informations de copyright
Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Déclaration de conflit d'intérêts
Declaration of interests The authors declare no competing interests.
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