title: Charge-pairing interactions control the conformational setpoint and motions of the FMN domain in neuronal nitric oxide synthase.
abstract: The NOS (nitric oxide synthase; EC 1.14.13.39) enzymes contain a C-terminal flavoprotein domain [NOSred (reductase domain of NOS)] that binds FAD and FMN, and an N-terminal oxygenase domain that binds haem. Evidence suggests that the FMN-binding domain undergoes large conformational motions to shuttle electrons between the NADPH/FAD-binding domain [FNR (ferredoxin NADP-reductase)] and the oxygenase domain. Previously we have shown that three residues on the FMN domain (Glu762, Glu816 and Glu819) that make charge-pairing interactions with the FNR help to slow electron flux through nNOSred (neuronal NOSred). In the present study, we show that charge neutralization or reversal at each of these residues alters the setpoint [Keq(A)] of the NOSred conformational equilibrium to favour the open (FMN-deshielded) conformational state. Moreover, computer simulations of the kinetic traces of cytochrome c reduction by the mutants suggest that they have higher conformational transition rates (1.5-4-fold) and rates of interflavin electron transfer (1.5-2-fold) relative to wild-type nNOSred. We conclude that the three charge-pairing residues on the FMN domain govern electron flux through nNOSred by stabilizing its closed (FMN-shielded) conformational state and by retarding the rate of conformational switching between its open and closed conformations.
authors.affiliation: Department of Pathobiology, Lerner Research Institute, The Cleveland Clinic, Cleveland, OH 44195, USA.
authors.forename: Mohammad Mahfuzul
authors.initials: MM
authors.lastname: Haque
authors.forename: Mekki
authors.initials: M
authors.lastname: Bayachou
authors.forename: Mohammed A
authors.initials: MA
authors.lastname: Fadlalla
authors.forename: Deborah
authors.initials: D
authors.lastname: Durra
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Stuehr
mesh_terms: D001665:Binding Sites / Q000235:genetics; D004579:Electron Transport; D005486:Flavin Mononucleotide / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D009068:Movement / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs / Q000235:genetics / Q000502:physiology*; D055672:Static Electricity
chemical_list: D005486:Flavin Mononucleotide; D052248:Nitric Oxide Synthase Type I; D009251:NADPH-Ferrihemoprotein Reductase