title: 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 FAD(-) 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.
authors.affiliation: Unité INSERM 696, Laboratory for Optics and Biosciences, Ecole Polytechnique, Palaiseau, France.
authors.forename: Edward
authors.initials: E
authors.lastname: Beaumont
authors.forename: Jean-Christophe
authors.initials: JC
authors.lastname: Lambry
authors.forename: Mireille
authors.initials: M
authors.lastname: Blanchard-Desce
authors.forename: Pavel
authors.initials: P
authors.lastname: Martasek
authors.forename: Satya P
authors.initials: SP
authors.lastname: Panda
authors.forename: Ernst E H
authors.initials: EE
authors.lastname: van Faassen
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Brochon
authors.forename: Eric
authors.initials: E
authors.lastname: Deprez
authors.forename: Anny
authors.initials: A
authors.lastname: Slama-Schwok
mesh_terms: D001665:Binding Sites; D055162:Biocatalysis; D002147:Calmodulin / Q000494:pharmacology; D004579:Electron Transport / Q000187:drug effects; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D009249:NADP / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis*; D052248:Nitric Oxide Synthase Type I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055668:Photochemical Processes; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence
chemical_list: D002147:Calmodulin; D005182:Flavin-Adenine Dinucleotide; D009569:Nitric Oxide; D009249:NADP; D052248:Nitric Oxide Synthase Type I