NO formation by neuronal NO-synthase can be controlled by ultrafast electron injection from a nanotrigger.
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.
Beaumont
Edward
E
Unité INSERM 696, Laboratory for Optics and Biosciences, Ecole Polytechnique, Palaiseau, France.
Lambry
Jean-Christophe
JC
Blanchard-Desce
Mireille
M
Martasek
Pavel
P
Panda
Satya P
SP
van Faassen
Ernst E H
EE
Brochon
Jean-Claude
JC
Deprez
Eric
E
Slama-Schwok
Anny
A
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
D002147:Calmodulin; D005182:Flavin-Adenine Dinucleotide; D009569:Nitric Oxide; D009249:NADP; D052248:Nitric Oxide Synthase Type I