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NO formation by neuronal NO-synthase can be controlled by ultrafast electron injection from a nanotrigger
Abstract : Nitric oxide synthases (NOSs) are unique flavohemoproteins with various roles in mammalian physiology. Constitutive NOS catalysis is initiated by fast hydride transfer from NADPH, followed by slower structural rearrangements. We used a photoactive nanotrigger (NT) to study the initial electron transfer to FAD in native neuronal NOS (nNOS) catalysis. Molecular modeling and fluorescence spectroscopy showed that selective NT binding to NADPH sites close to FAD is able to override Phe1395 regulation. Ultrafast injection of electrons into the protein electron pathway by NT photoactivation through the use of a femtosecond laser pulse is thus possible. We show that calmodulin, required for NO synthesis by constitutive NOS, strongly promotes intramolecular electron flow (6.2-fold stimulation) by a mechanism involving proton transfer to the reduced FADb site. Site-directed mutagenesis using the S1176A and S1176T mutants of nNOS supports this hypothesis. The NT synchronized the initiation of flavoenzyme catalysis, leading to the formation of NO, as detected by EPR. This NT is thus promising for time-resolved X-ray and other cellular applications. Cop. 2009 Wiley-VCH Verlag GmbH and Co. KGaA, Weinheim.
Keywords :
ELECTRON INJECTION
NANOTRIGGER
https://hal-polytechnique.archives-ouvertes.fr/hal-00818490
Déposant : Denis Roura <>
Soumis le : lundi 6 mai 2013 - 21:43:10
Dernière modification le : jeudi 4 mars 2021 - 16:37:14
Déposant : Denis Roura <>
Soumis le : lundi 6 mai 2013 - 21:43:10
Dernière modification le : jeudi 4 mars 2021 - 16:37:14
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