Dataset Information

Control of electron transfer and catalysis in neuronal nitric-oxide synthase (nNOS) by a hinge connecting its FMN and FAD-NADPH domains.

ABSTRACT: In nitric-oxide synthases (NOSs), two flexible hinges connect the FMN domain to the rest of the enzyme and may guide its interactions with partner domains for electron transfer and catalysis. We investigated the role of the FMN-FAD/NADPH hinge in rat neuronal NOS (nNOS) by constructing mutants that either shortened or lengthened this hinge by 2, 4, and 6 residues. Shortening the hinge progressively inhibited electron flux through the calmodulin (CaM)-free and CaM-bound nNOS to cytochrome c, whereas hinge lengthening relieved repression of electron flux in CaM-free nNOS and had no impact or slowed electron flux through CaM-bound nNOS to cytochrome c. How hinge length influenced heme reduction depended on whether enzyme flavins were pre-reduced with NADPH prior to triggering heme reduction. Without pre-reduction, changing the hinge length was deleterious; with pre-reduction, the hinge shortening was deleterious, and hinge lengthening increased heme reduction rates beyond wild type. Flavin fluorescence and stopped-flow kinetic studies on CaM-bound enzymes suggested hinge lengthening slowed the domain-domain interaction needed for FMN reduction. All hinge length changes lowered NO synthesis activity and increased uncoupled NADPH consumption. We conclude that several aspects of catalysis are sensitive to FMN-FAD/NADPH hinge length and that the native hinge allows a best compromise among the FMN domain interactions and associated electron transfer events to maximize NO synthesis and minimize uncoupled NADPH consumption.

SUBMITTER: Haque MM

PROVIDER: S-EPMC3436266 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Control of electron transfer and catalysis in neuronal nitric-oxide synthase (nNOS) by a hinge connecting its FMN and FAD-NADPH domains.

Haque Mohammad Mahfuzul MM Fadlalla Mohammed A MA Aulak Kulwant S KS Ghosh Arnab A Durra Deborah D Stuehr Dennis J DJ

The Journal of biological chemistry 20120620 36

In nitric-oxide synthases (NOSs), two flexible hinges connect the FMN domain to the rest of the enzyme and may guide its interactions with partner domains for electron transfer and catalysis. We investigated the role of the FMN-FAD/NADPH hinge in rat neuronal NOS (nNOS) by constructing mutants that either shortened or lengthened this hinge by 2, 4, and 6 residues. Shortening the hinge progressively inhibited electron flux through the calmodulin (CaM)-free and CaM-bound nNOS to cytochrome c, wher ...[more]

PMID: 22722929

Similar Datasets

Project description:In our previous studies, CB(1) cannabinoid receptor agonists stimulated production of cyclic GMP and translocation of nitric oxide (NO)-sensitive guanylyl cyclase in neuronal cells (Jones et al., Neuropharmacology 54:23-30, 2008). The purpose of these studies was to elucidate the signal transduction of cannabinoid-mediated neuronal nitric oxide synthase (nNOS) activation in neuronal cells. Cannabinoid agonists CP55940 (2-[(1S,2R,5S)-5-hydroxy-2-(3-hydroxypropyl) cyclohexyl]-5-(2-methyloctan-2-yl)phenol), WIN55212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate), and the metabolically stable analog of anandamide, (R)-(+)-methanandamide stimulated NO production in N18TG2 cells over a 20-min period. Rimonabant (N-(piperidin-lyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-H-pyrazole-3-carboxamide), a CB(1) receptor antagonist, partially or completely curtailed cannabinoid-mediated NO production. Inhibition of NOS activity (N ( G )-nitro-L: -arginine) or signaling via Gi/o protein (pertussis toxin) significantly limited NO production by cannabinoid agonists. Ca(2+) mobilization was not detected in N18TG2 cells after cannabinoid treatment using Fluo-4 AM fluorescence. Cannabinoid-mediated NO production was attributed to nNOS activation since endothelial NOS and inducible NOS protein and mRNA were not detected in N18TG2 cells. Bands of 160 and 155 kDa were detected on Western blot analysis of cytosolic and membrane fractions of N18TG2 cells, using a nNOS antibody. Chronic treatment of N18TG2 cells with cannabinoid agonists downregulated nNOS protein and mRNA as detected using Western blot analysis and real-time polymerase chain reaction, respectively. Cannabinoid agonists stimulated NO production via signaling through CB(1) receptors, leading to activation of Gi/o protein and enhanced nNOS activity. The findings of these studies provide information related to cannabinoid-mediated NO signal transduction in neuronal cells, which has important implications in the ongoing elucidation of the endocannabinoid system in the nervous system.

Project description:Nitric oxide (NO) is a neurotransmitter synthesized in the brain by neuronal nitric oxide synthase (nNOS). Using immunohistochemistry and confocal imaging in the inferior colliculus (IC, auditory midbrain) of the guinea pig (Cavia porcellus, male and female), we show that nNOS occurs in two distinct cellular distributions. We confirm that, in the cortices of the IC, a subset of neurons show cytoplasmic labeling for nNOS, whereas in the central nucleus (ICc), such neurons are not present. However, we demonstrate that all neurons in the ICc do in fact express nNOS in the form of discrete puncta found at the cell membrane. Our multi-labeling studies reveal that nNOS puncta form multiprotein complexes with NMDA receptors, soluble guanylyl cyclase (sGC), and PSD95. These complexes are found apposed to glutamatergic terminals, which is indicative of synaptic function. Interestingly, these glutamatergic terminals express both vesicular glutamate transporters 1 and 2 denoting a specific source of brainstem inputs. With in vivo electrophysiological recordings of multiunit activity in the ICc, we found that local application of NMDA enhances sound-driven activity in a concentration-dependent and reversible fashion. This response is abolished by blockade of nNOS or sGC, indicating that the NMDA effect is mediated solely via the NO and cGMP signaling pathway. This discovery of a ubiquitous, but highly localized, expression of nNOS throughout the ICc and demonstration of the dramatic influence of the NMDA activated NO pathway on sound-driven neuronal activity imply a key role for NO signaling in auditory processing.SIGNIFICANCE STATEMENT We show that neuronal nitric oxide synthase (nNOS), the enzyme that synthesizes nitric oxide (NO), occurs as puncta in apparently all neurons in the central nucleus of the inferior colliculus (ICc) in the auditory midbrain. Punctate nNOS appears at glutamatergic synapses in a complex with glutamate NMDA receptors (NMDA-Rs), soluble guanylyl cyclase (sGC, the NO receptor), and PSD95 (a protein that anchors receptors and enzymes at the postsynaptic density). We show that NMDA-R modulation of sound-driven activity in the ICc is solely mediated by activation of nNOS and sGC. The presence of nNOS throughout this sensory nucleus argues for a major role of NO in hearing. Furthermore, this punctate form of nNOS expression may exist and have gone unnoticed in other brain regions.

Dataset Information

Control of electron transfer and catalysis in neuronal nitric-oxide synthase (nNOS) by a hinge connecting its FMN and FAD-NADPH domains.

Publications

Control of electron transfer and catalysis in neuronal nitric-oxide synthase (nNOS) by a hinge connecting its FMN and FAD-NADPH domains.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets