TY - JOUR
T1 - NO formation by neuronal NO-synthase can be controlled by ultrafast electron injection from a nanotrigger
AU - Beaumont, Edward
AU - Lambry, Jean Christophe
AU - Blanchard-Desce, Mireille
AU - Martasek, Pavel
AU - Panda, Satya P.
AU - van Faassen, Ernst E.H.
AU - Brochon, Jean Claude
AU - Deprez, Eric
AU - Slama-Schwok, Anny
N1 - Copyright:
Copyright 2018 Elsevier B.V., All rights reserved.
PY - 2009/3/2
Y1 - 2009/3/2
N2 - 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.
AB - 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.
KW - Emission spectroscopy
KW - Flavins
KW - Kinetics
KW - Metalloenzymes
KW - Molecular modeling
KW - Photocatalysis
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U2 - 10.1002/cbic.200800721
DO - 10.1002/cbic.200800721
M3 - Article
C2 - 19222033
AN - SCOPUS:65549083804
VL - 10
SP - 690
EP - 701
JO - ChemBioChem
JF - ChemBioChem
SN - 1439-4227
IS - 4
ER -