Brain nitric oxide synthase is a biopterin‐ and flavin‐containing multi‐functional oxido‐reductase

Mayer, Bernd; John, Mathias; Heinzel, Burghard; Werner, Ernst R.; Wächter, Helmut; Schultz, Günter; Böhme, Eycke

doi:10.1016/0014-5793(91)81031-3

Cited by 404 publications

(225 citation statements)

References 34 publications

(7 reference statements)

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“…Potential post-synthetic reactions could be traced and some of the proposals made concerning the identity of EDRF could be excluded, if one could be sure that NO is the immediate product generated by NO synthases. -Unfortunately, this is questioned [34][35][36]49], and the molecular mechanism of the NO synthase reaction(s) still needs to be clarified [31][32][33]. We now demonstrate for the first time that NO derived from the guanidino nitrogen ofL-arginine accumulates stoichiometrically with respect to L-citrulline in cell-free incubation mixtures of endothelial and macrophage NO synthases (experiments with [l5N]NG-L-arginine).…”

Section: Discussionmentioning

confidence: 96%

“…generated by the NO synthase reaction remained unknown, as until now the molecular mechanism of this reaction has not been established [31][32][33]. Recently spintrapping experiments identified a carbon-centred radical in lipid extracts from acetylcholine-stimulated arteries [34] in addition to an unidentified radical in cytosol from L-arginine-supplemented neuroblastoma cells [35] and platelets [36].…”

Section: Introductionmentioning

confidence: 99%

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NO accounts completely for the oxygenated nitrogen species generated by enzymic l-arginine oxygenation

Mülsch

Vanin

Mordvintcev

et al. 1992

Biochemical Journal

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Section: Discussionmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

NO accounts completely for the oxygenated nitrogen species generated by enzymic l-arginine oxygenation

Mülsch

Vanin

Mordvintcev

et al. 1992

Biochemical Journal

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“…Incubations were for 10 min at 37°C in 0.1 ml of 50 mM triethanolamine/HCl buffer (pH 7.4) containing 0.1 mM L- [2,3,4,5-3 H]arginine (ϳ80,000 counts/min), 0.5 mM CaCl 2 , 10 g/ml CaM, 0.2 mM NADPH, 10 M BH 4 , 5 M FAD, 5 M FMN, 0.2 mM CHAPS. CaM-dependent NADPH:oxygen and NADPH:cytochrome c oxidoreductase activities of nNOS and eNOS were determined spectrophotometrically in the absence of L-arginine as described previously (26,27).…”

Section: Methodsmentioning

confidence: 99%

Interaction of Endothelial and Neuronal Nitric-oxide Synthases with the Bradykinin B2 Receptor

Golser¹,

Gorren²,

Leber³

et al. 2000

Journal of Biological Chemistry

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Endothelial nitric-oxide synthase (type III) (eNOS) was reported to form an inhibitory complex with the bradykinin receptor B2 (B2R) from which the enzyme is released in an active form upon receptor activation (Ju, H., Venema, V. J., Marrero, M. B., and Venema, R. C. (1998) J. Biol. Chem. 273, 24025-24029). Using a synthetic peptide derived from the known inhibitory sequence of the B2R (residues 310 -329) we studied the interaction of the receptor with purified eNOS and neuronal nitricoxide synthase (type I) (nNOS). The peptide inhibited formation of L-citrulline by eNOS and nNOS with IC 50 values of 10.6 ؎ 0.4 M and 7.1 ؎ 0.6 M, respectively. Inhibition was not due to an interference of the peptide with L-arginine or tetrahydrobiopterin binding. The NADPH oxidase activity of nNOS measured in the absence of L-arginine was inhibited by the peptide with an IC 50 of 3.7 ؎ 0.6 M, but the cytochrome c reductase activity of the enzyme was much less susceptible to inhibition (IC 50 >0.1 mM). Steady-state absorbance spectra of nNOS recorded during uncoupled NADPH oxidation showed that the heme remained oxidized in the presence of the synthetic peptide consisting of amino acids 310 -329 of the B2R, whereas the reduced oxyferrous heme complex was accumulated in its absence. These data suggest that binding of the B2R 310 -329 peptide blocks flavin to heme electron transfer. Co-immunoprecipitation of B2R and nNOS from human embryonic kidney cells stably transfected with human nNOS suggests that the B2R may functionally interact with nNOS in vivo. This interaction of nNOS with the B2R may recruit the enzyme to allow for the effective coupling of bradykinin signaling to the nitric oxide pathway. Nitric oxide (NO)1 is a signaling molecule that is synthesized from L-arginine and O 2 by nitric-oxide synthases (NOS, EC 1.14.13.39) that exist in the three following isozymes (for a review see Refs. 1 and 2): nNOS (type I), eNOS (type III), and inducible NOS (type II). nNOS and eNOS are constitutively expressed and Ca 2ϩ -dependent, whereas inducible NOS is cytokine-inducible and Ca 2ϩ -independent. All three isozymes are homodimers, with each subunit consisting of two domains, an amino-terminal oxygenase domain containing a heme group and binding sites for the substrate L-arginine and the cofactor BH 4 and a carboxyl-terminal reductase domain with binding sites for NADPH and the flavins FAD and FMN. The two domains are joined by a binding region for CaM that has a dual function, activating electron transfer both from NADPH to the flavins and from the reductase to the oxygenase domain (3). Although inducible NOS binds CaM in nominally Ca 2ϩ -free solutions (Ͻ30 nM), free Ca 2ϩ in the micromolar range is essential for CaM binding to the constitutive isozymes, so that a rise in intracellular free Ca 2ϩ is a key signal for ligand-induced activation of nNOS and eNOS (for review see Ref. 1). In vascular endothelial cells, fluid shear stress triggers an alternative, apparently Ca 2ϩ -independent pathway of eNOS activation (for review see ...

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“…The reductase domains in eukaryotic NOS are homologous to NADPH P450 reductase (11). These domains contain one flavin mononucleotide (FMN) and one flavin adenine dinucleotide (FAD) cofactor (11-18), which function in electron transfer from NADPH to the heme-containing active site (13,17,19,20).Ca 2ϩ /CaM control in NOS is exerted through the regulation of electron transfer between NADPH and heme (5). This regulation is primarily a function of the reductase domains (21).…”

mentioning

confidence: 99%

mentioning

confidence: 99%

The Function of the Small Insertion in the Hinge Subdomain in the Control of Constitutive Mammalian Nitric-oxide Synthases

Jones

Smith

Gao

et al. 2004

Journal of Biological Chemistry

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Control of nitric oxide (NO) synthesis in the constitutive nitric-oxide synthases (NOS) by calcium/calmodulin is exerted through the regulation of electron transfer from NADPH through the reductase domains. This process has been shown previously to involve the calmodulin binding site, the autoinhibitory insertion in the FMN binding domain, and the C-terminal tail. Smaller sequence elements also appear to correlate with control. Although some of these elements appear well positioned to function in control, they are poorly conserved; their role in control is neither well established nor defined by available information. In this study mutations have been induced in the small insertion of the hinge subdomain, which has been shown recently to form a ␤ hairpin in structural studies of the neuronal NOS reductase domains adjacent to the calmodulin site and the autoinhibitory element. Modification of the small insertion in neuronal NOS tends to increase cytochrome c reduction but not NO synthetic activity; some modifications or deletions in the corresponding region in endothelial NOS modestly increase activity under some conditions. Unexpectedly, some minor changes in the sequence introduce a loss in the content of heme relative to flavin cofactors. Taken together, these results suggest that the small insertion protects the calmodulin binding site and that it may be a modulator of NOS activity.Nitric-oxide synthases (NOS) 1 are a growing family of modular enzymes, including three mammalian isoforms, as well as a number of related eukaryotic and more distantly related prokaryotic enzymes. Because of the discovery of the function of nitric oxide (NO) as a molecular messenger in a variety of signal transduction pathways (1-4), the endothelial and neuronal isoforms (eNOS and nNOS) have been shown to function as signal generators controlled by calcium/calmodulin (Ca 2ϩ / CaM) (5). The other mammalian isoform, iNOS, is induced during immune response (6 -9); it is constitutively active and binds CaM at basal Ca 2ϩ levels.NO synthesis from arginine and molecular oxygen requires 3 mol of electrons supplied by NADPH/mol of NO formed (10). The reductase domains in eukaryotic NOS are homologous to NADPH P450 reductase (11). These domains contain one flavin mononucleotide (FMN) and one flavin adenine dinucleotide (FAD) cofactor (11-18), which function in electron transfer from NADPH to the heme-containing active site (13,17,19,20).Ca 2ϩ /CaM control in NOS is exerted through the regulation of electron transfer between NADPH and heme (5). This regulation is primarily a function of the reductase domains (21). The canonical CaM binding site was recognized in the sequence region connecting the oxygenase and reductase regions (11). A major insertion in the FMN binding region, correlating with Ca 2ϩ /CaM control, was identified as a control element with autoinhibitory character (autoinhibitory insertion, or AI) (22,23). This has been confirmed by a number of groups (e.g. 24,25).Recently the C-terminal region has been shown to be necessary ...

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Brain nitric oxide synthase is a biopterin‐ and flavin‐containing multi‐functional oxido‐reductase

Cited by 404 publications

References 34 publications

NO accounts completely for the oxygenated nitrogen species generated by enzymic l-arginine oxygenation

NO accounts completely for the oxygenated nitrogen species generated by enzymic l-arginine oxygenation

Interaction of Endothelial and Neuronal Nitric-oxide Synthases with the Bradykinin B2 Receptor

The Function of the Small Insertion in the Hinge Subdomain in the Control of Constitutive Mammalian Nitric-oxide Synthases

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