The mechanism of superoxide generation by endothelial nitric oxide synthase (eNOS) was investigated by the electron spin resonance spin-trapping technique using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide. In the absence of calcium͞calmodulin, eNOS produces low amounts of superoxide. Upon activating eNOS electron transfer reactions by calcium͞calmodulin binding, superoxide formation is increased. Heme-iron ligands, cyanide, imidazole, and the phenyl(diazene)-derived radical inhibit superoxide generation. No inhibition is observed after addition of L-arginine, N G -hydroxy-L-arginine, L-thiocitrulline, and L-N G -monomethyl arginine to activated eNOS. These results demonstrate that superoxide is generated from the oxygenase domain by dissociation of the ferrous-dioxygen complex and that occupation of the L-arginine binding site does not inhibit this process. However, the concomitant addition of L-arginine and tetrahydrobiopterin (BH 4 ) abolishes superoxide generation by eNOS. Under these conditions, L-citrulline production is close to maximal. Our data indicate that BH 4 fully couples L-arginine oxidation to NADPH consumption and prevents dissociation of the ferrous-dioxygen complex. Under these conditions, eNOS does not generate superoxide. The presence of f lavins, at concentrations commonly employed in NOS assay systems, enhances superoxide generation from the reductase domain. Our data indicate that modulation of BH 4 concentration may regulate the ratio of superoxide to nitric oxide generated by eNOS.

Nitric oxide, a key signaling molecule, is produced by a family of enzymes collectively called nitric oxide synthases (NOS). Here, we report the crystal structure of the heme domain of endothelial NOS in tetrahydrobiopterin (H4B)-free and -bound forms at 1.95 A and 1.9 A resolution, respectively. In both structures a zinc ion is tetrahedrally coordinated to pairs of symmetry-related cysteine residues at the dimer interface. The phylogenetically conserved Cys-(X)4-Cys motif and its strategic location establish a structural role for the metal center in maintaining the integrity of the H4B-binding site. The unexpected recognition of the substrate, L-arginine, at the H4B site indicates that this site is poised to stabilize a positively charged pterin ring and suggests a model involving a cationic pterin radical in the catalytic cycle.

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The neuronal nitric oxide synthase (nNOS) has been successfully overexpressed in Escherichia coli, with average yields of 125-150 nmol (20-24 mg) of enzyme per liter of cells. The cDNA for nNOS was subcloned into the pCW vector under the control of the tac promotor and was coexpressed with the chaperonins groEL and groES in the protease-deficient BL21 strain ofE. coli. The enzyme produced is replete with heme and flavins and, after overnight incubation with tetrahydrobiopterin, contains 0.7 pmol of tetrahydrobiopterin per pmol of nNOS. nNOS is isolated as a predominantly high-spin heme protein and demonstrates spectral properties that are identical to those of nNOS isolated from stably transfected human kidney 293 cells. It binds N'0-nitroarginine dependent on the presence of bound tetrahydrobiopterin and exhibits a Kd of 45 nM. The enzyme is completely functional; the specific activity is 450 nmol/min per mg. This overexpression system will be extremely useful for rapid, inexpensive preparation of large amounts of active nNOS for use in mechanistic and structure/function studies, as well as for drug design and development.

Nitric oxide synthases (NOSs) are classified functionally, based on whether calmodulin binding is Ca 2؉ -dependent (cNOS) or Ca 2؉ -independent (iNOS). This key dichotomy has not been defined at the molecular level. Here we show that cNOS isoforms contain a unique polypeptide insert in their FMN binding domains which is not shared with iNOS or other related flavoproteins. Previously identified autoinhibitory domains in calmodulin-regulated enzymes raise the possibility that the polypeptide insert is the autoinhibitory domain of cNOSs. Consistent with this possibility, three-dimensional molecular modeling suggested that the insert originates from a site immediately adjacent to the calmodulin binding sequence. Synthetic peptides derived from the 45-amino acid insert of endothelial NOS were found to potently inhibit binding of calmodulin and activation of cNOS isoforms. This inhibition was associated with peptide binding to NOS, rather than free calmodulin, and inhibition could be reversed by increasing calmodulin concentration. In contrast, insert-derived peptides did not interfere with the arginine site of cNOS, as assessed from [ 3 H]N G -nitro-L-arginine binding, nor did they potently effect iNOS activity. Limited proteolysis studies showed that calmodulin's ability to gate electron flow through cNOSs is associated with displacement of the insert polypeptide; this is the first specific calmodulin-induced change in NOS conformation to be identified. Together, our findings strongly suggest that the insert is an autoinhibitory control element, docking with a site on cNOSs which impedes calmodulin binding and enzymatic activation. The autoinhibitory control element molecularly defines cNOSs and offers a unique target for developing novel NOS activators and inhibitors.Nitric oxide is a ubiquitous cell-signaling molecule, with protean roles in physiology and pathophysiology (1-3). Encoded by distinct genes, mammalian NO synthases (NOSs) 1 comprise a family of three calmodulin-dependent biopterohemoflavoproteins that are functionally distinguished by their modes of regulation (4). The two constitutively expressed isoforms of NOS (cNOSs), first identified in neuronal cells (nNOS) and endothelial cells (eNOS), remain dormant until calcium/calmodulin (Ca 2ϩ /CaM) binding is actuated by transient elevations in intracellular Ca 2ϩ . This Ca 2ϩ -dependent mode of regulation provides pulses of NO for moment-to-moment modulation of vascular tone and neurosignaling. In contrast, activity of the immunostimulant-induced isoform of NOS (iNOS) is Ca 2ϩ -independent, providing continuous high output NO generation for host defense. A remarkably high affinity for CaM, even at basally low levels of intracellular calcium, is responsible for the Ca 2ϩ independence of iNOS (5). Whether a given NOS isoform binds CaM in a Ca 2ϩ -dependent or -independent manner has been assumed to be a property solely of the amino acid sequence specified by a 20 -25-amino acid CaM binding site. However, this restrictive view is challenged by findings that ...

Endothelial nitric oxide synthase (eNOS) is a dually acylated peripheral membrane protein that targets to the Golgi region and caveolae of endothelial cells. Recent evidence has shown that eNOS can co-precipitate with caveolin-1, the resident coat protein of caveolae, suggesting a direct interaction between these two proteins. To test this idea, we examined the interactions of eNOS with caveolin-1 in vitro and in vivo. Incubation of endothelial cell lysates or purified eNOS with glutathione S-transferase (GST)-caveolin-1 resulted in the direct interaction of the two proteins. Utilizing a series of GSTcaveolin-1 deletion mutants, we identified two cytoplasmic domains of caveolin-1 that interact with eNOS, the scaffolding domain (amino acids 61-101) and to a lesser extent the C-terminal tail (amino acids 135-178). Incubation of pure eNOS with peptides derived from the scaffolding domains of caveolin-1 and -3, but not the analogous regions from caveolin-2, resulted in inhibition of eNOS, inducible NOS (iNOS), and neuronal NOS (nNOS) activities. These results suggest a common mechanism and site of inhibition. Utilizing GST-eNOS fusions, the site of caveolin binding was localized between amino acids 310 and 570. Site-directed mutagenesis of the predicted caveolin binding motif within eNOS blocked the ability of caveolin-1 to suppress NO release in co-transfection experiments. Thus, our data demonstrate a novel functional role for caveolin-1 in mammalian cells as a potential molecular chaperone that directly inactivates NOS. This suggests that the direct binding of eNOS to caveolin-1, per se, and the functional consequences of eNOS targeting to caveolae are likely temporally and spatially distinct events that regulate NO production in endothelial cells. Additionally, the inactivation of eNOS and nNOS by the scaffolding domain of caveolin-3 suggests that eNOS in cardiac myocytes and nNOS in skeletal muscle are likely subject to negative regulation by this muscle-specific caveolin isoform.Caveolae are cholesterol-and sphingolipid-rich microdomains of the plasmalemma that have been implicated in a variety of cellular functions, including transcytosis of molecules and signal transduction events (1, 2). With respect to the latter function, structurally distinct dually acylated proteins involved in signal transduction (including G-protein ␣ subunits, Ha-Ras, Src family members, and endothelial nitric oxide synthase (eNOS) 1 ) reside in caveolae (3-6). In the case of certain Src members and eNOS, mutation of the cysteine palmitoylation sites prevents caveolae localization suggesting that palmitoylation is a "molecular zip code" for the trafficking of dually acylated proteins into glycolipid-rich microdomains of the plasmalemma (4, 6, 7).The major coat proteins of caveolae are the caveolin family of proteins (caveolin-1, -2 and -3 (1, 8)). Besides being intimately embedded within the lipid microdomain comprising caveolae, caveolins may regulate signaling via direct interaction with other resident proteins. For example, caveol...

The sequences of nitric-oxide synthase flavin domains closely resemble that of NADPH-cytochrome P450 reductase (CPR). However, all nitric-oxide synthase (NOS) isoforms are 20 -40 residues longer in the C terminus, forming a "tail" that is absent in CPR. To investigate its function, we removed the 33 and 42 residue C termini from neuronal NOS (nNOS) and endothelial NOS (eNOS), respectively. Both truncated enzymes exhibited cytochrome c reductase activities without calmodulin that were 7-21-fold higher than the nontruncated forms. With calmodulin, the truncated and wild-type enzymes reduced cytochrome c at approximately equal rates. Therefore, calmodulin functioned as a nonessential activator of the wild-type enzymes and a partial noncompetitive inhibitor of the truncated mutants. Truncated nNOS and eNOS plus calmodulin catalyzed NO formation at rates that were 45 and 33%, respectively, those of their intact forms. Without calmodulin, truncated nNOS and eNOS synthesized NO at rates 14 and 20%, respectively, those with calmodulin. By using stopped-flow spectrophotometry, we demonstrated that electron transfer into and between the two flavins is faster in the absence of the C terminus. Although both CPR and intact NOS can exist in a stable, one-electron-reduced semiquinone form, neither of the truncated enzymes do so. We propose negative modulation of FAD-FMN interaction by the C termini of both constitutive NOSs.Nitric-oxide synthases (NOSs) 1 are bidomain, dimeric enzymes that synthesize NO from L-arginine through a series of monooxygenation reactions (for review see Ref. 1). The three isoforms, neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS), produce NO by the same mechanism but play very different physiological roles due to the type of cell where they are located. nNOS, located in neurons in the brain and at neuromuscular junctions, is involved in neurotransmission (2, 3); iNOS, located in macrophages, is involved in the immune response (4, 5); and eNOS, located in endothelial cells, is involved in hemodynamic regulation (6, 7). The NO produced by nNOS and eNOS exerts its effects through the stimulation of guanylate cyclase, whereas the NO produced by iNOS exerts its effects directly or by combining with superoxide anion radical to form peroxynitrite anion, both potent oxidants deleterious to proteins and DNA.The NOSs consist of two domains, a heme and H 4 B-containing oxygenase (or heme) domain, which binds the substrate L-arginine, and a flavin-containing reductase (or flavoprotein) domain, which binds the prosthetic group flavins FAD and FMN and the cofactor NADPH. Electrons are transferred into NOS at the FAD moiety and are subsequently passed through the FMN to the heme domain. A calmodulin-binding region bisects the two domains. Calmodulin (CaM) is required for NO production and mediates the transfer of electrons from the FMN of NOS to the heme domain (8). CaM binds NOS in a 1:1 stoichiometry with very high affinity. CaM binds to sequences within the nNOS, eNOS, and iNOS CaM-binding...

The nitric oxide synthases (NOS-I, neuronal, NOS-II, inducible, and NOS-III, endothelial) are the most recent additions to the large number of heme proteins that contain cysteine thiolate-liganded protoporphyrin IX heme prosthetic groups. This group of oxygenating enzymes also includes one of the largest gene families, that of the cytochromes P450, which have been demonstrated to be involved in the hydroxylation of a variety of substrates, including endogenous compounds (steroids, fatty acids, and prostaglandins) and exogenous compounds (therapeutic drugs, environmental toxicants, and carcinogens). The substrates for cytochromes P450 are universally hydrophobic while the physiological substrate for the nitric oxide synthases is the amino acid L-arginine, a hydrophilic compound. This review will discuss the approaches being used to study the structure and mechanism of neuronal nitric oxide synthase in the context of its known prosthetic groups and regulation by Ca(2+)-calmodulin and/or tetrahydrobiopterin (BH4).