The ferrous dioxygen complex of the oxygenase domain of neuronal nitric-oxide synthase

The mechanisms by which nitric-oxide synthases (NOSs) bind and activate oxygen at their P450-type heme active site in order to synthesize nitric oxide from the substrate L-arginine are mostly unknown. To obtain information concerning the structure and properties of the first oxygenated intermediate of the enzymatic cycle, we have used a rapid continuous flow mixer and resonance Raman spectroscopy to generate and identify the ferrous dioxygen complex of the oxygenase domain of nNOS (Fe(2+)O(2) nNOSoxy). We detect a line at 1135 cm(-1) in the resonance Raman spectrum of the intermediate formed from 0.6 to 3.0 ms after the rapid mixing of the ferrous enzyme with oxygen that is shifted to 1068 cm(-1) with (18)O(2). This line is assigned as the O-O stretching mode (nu(O-O)) of the oxygenated complex of nNOSoxy. Rapid mixing experiments performed with nNOSoxy saturated with L-arginine or N(omega)-hydroxy-L-arginine, in the presence or absence of (6R)-5,6, 7,8-tetrahydro-L-biopterin, reveal that the nu(O-O) line is insensitive to the presence of the substrate and the pterin. The optical spectrum of this ferrous dioxygen species, with a Soret band wavelength maximum at 430 nm, confirms the identification of the previously reported oxygenated complexes generated by stopped flow techniques.     

Interaction of neuronal nitric-oxide synthase and phosphofructokinase-M

Neurons that express neuronal nitric-oxide synthase (nNOS) are resistant to NO-induced neurotoxicity; however, the mechanism by which these neurons are protected is not clear. To identify proteins possibly involved in this process, we performed affinity chromatography with the nNOS PDZ domain, a N-terminal motif that mediates protein interactions. Using this method to fractionate soluble tissue extracts, we identified the muscle isoform of phosphofructokinase (PFK-M) as a protein that binds to nNOS both in brain and skeletal muscle. PFK-M interacts with the PDZ domain of nNOS, and nNOS-PFK-M binding can be competed by peptides that bind to the PDZ domain of nNOS. We found that nNOS is significantly associated with PFK-M in skeletal muscle because nNOS can be immunodepleted from cytosolic skeletal muscle extracts using an antibody directed against PFK-M. In brain, nNOS and PFK-M are both enriched in synaptosomes, and specifically, in the synaptic vesicle fraction, where they can interact. At the cellular level, PFK-M is enriched in neurons that express nNOS protein. As fructose-1, 6-bisphosphate, the product of PFK activity, is neuroprotective, the interaction of nNOS and PFK may contribute to neuroprotection of nNOS positive cells.     

L-type voltage-operated calcium channels, N-methyl-D-aspartate receptors and neuronal nitric-oxide synthase form a calcium/redox nano-transducer within lipid rafts

Cytosolic calcium plays a leading role in the control of neuronal excitability, plasticity and survival. This work aims to experimentally assess the possibility that lipid rafts of the plasma membrane can provide a structural platform for a faster and tighter functional coupling between calcium and nitric-oxide signaling in neurons. Using primary cerebellar granule neurons (CGN) in culture this hypothesis has been experimentally assessed with fluorescence resonance energy transfer imaging, preparations of lipid rafts-enriched membrane fragments and western blotting. The results obtained in this work demonstrated that major calcium entry systems of the plasma membrane of CGN (L-type calcium channels and N-methyl-D-aspartate receptors) and nitric-oxide synthase are separated by less than 80 nm from each other within lipid rafts-associated sub-microdomains, suggesting a new role of lipid rafts as neuronal calcium/redox nano-transducers.     

Aminoguanidine-mediated inactivation and alteration of neuronal nitric-oxide synthase

It is established that aminoguanidine (AG) is a metabolism-based inactivator of the three major isoforms of nitric-oxide synthase. AG is thought to be of potential use in diseases, such as diabetes, where pathological overproduction of NO is implicated. We show here that during the inactivation of neuronal nitric-oxide synthase (nNOS) by AG that the prosthetic heme is altered, in part, to dissociable and protein-bound adducts. The protein-bound heme adduct is the result of cross-linking of the heme to residues in the oxygenase domain of nNOS. The dissociable heme product is unstable and reverts back to heme upon isolation. The alteration of the heme is concomitant with the loss in the ability to form the ferrous-CO complex of nNOS and accounts for at least two-thirds of the activity loss. Studies with [(14)C]AG indicate that alteration of the protein, in part on the reductase domain of nNOS, also occurs but at low levels. Thus, heme alteration appears to be the major cause of nNOS inactivation. The elucidation of the mechanism of inactivation of nNOS will likely lead to a better understanding of the in vivo effects of NOS inhibitors such as AG.     

Inhibition of neuronal nitric-oxide synthase by calcium/ calmodulin-dependent protein kinase IIalpha through Ser847 phosphorylation in NG108-15 neuronal cells

We have previously demonstrated that phosphorylation of neuronal nitric-oxide synthase (nNOS) at Ser(847) by Ca(2+)/calmodulin-dependent protein kinases (CaM kinases) attenuates the catalytic activity of the enzyme in vitro (Hayashi Y., Nishio M., Naito Y., Yokokura H., Nimura Y., Hidaka H., and Watanabe Y. (1999) J. Biol. Chem. 274, 20597-20602). In the present study we determined that CaM kinase IIalpha (CaM-K IIalpha) can directly phosphorylate nNOS on Ser(847), leading to a reduction of nNOS activity in cells. The phosphorylation abilities of purified CaM kinase Ialpha (CaM-K Ialpha), CaM-K IIalpha, and CaM-kinase IV (CaM-K IV) on Ser(847) were analyzed using the synthetic peptide nNOS-(836-859) (Glu-Glu-Arg-Lys-Ser-Tyr-Lys-Val-Arg-Phe-Asn-Ser-Val-Ser-Ser-Tyr-Ser- Asp-Ser-Arg-Lys-Ser-Ser-Gly) from nNOS as substrate. The relative V(max)/K(m) ratios of CaM kinases for nNOS-(836-859) were found to be as follows: CaM-K IIalpha, 100; CaM-K Ialpha, 54.5; CaM-K IV, 9.1. Co-transfection of constitutively active CaM-K IIalpha1-274 but not inactive CaM-K IIalpha1-274, generated by mutation of Lys(42) to Ala, with nNOS into NG108-15 cells, resulted in increased Ser(847) phosphorylation in the presence of okadaic acid, an inhibitor of protein phosphatase (PP)1 and PP2A, with a concomitant inhibition of NOS enzyme activity. In addition, this latter decrease could be reversed by treatment with exogenous PP2A. Cells expressing mutant nNOS (S847A) proved resistant to phosphorylation and a decrease of NOS activity. Thus, our results indicate that Ca(2+) triggers cross-talk signal transduction between CaM kinase and NO and CaM-K IIalpha phosphorylating nNOS on Ser(847), which in turn decreases the gaseous second messenger NO in neuronal cells.     

Plasma membrane calcium pump (PMCA4)-neuronal nitric-oxide synthase complex regulates cardiac contractility through modulation of a compartmentalized cyclic nucleotide microdomain

Identification of the signaling pathways that regulate cyclic nucleotide microdomains is essential to our understanding of cardiac physiology and pathophysiology. Although there is growing evidence that the plasma membrane Ca(2+)/calmodulin-dependent ATPase 4 (PMCA4) is a regulator of neuronal nitric-oxide synthase, the physiological consequence of this regulation is unclear. We therefore tested the hypothesis that PMCA4 has a key structural role in tethering neuronal nitric-oxide synthase to a highly compartmentalized domain in the cardiac cell membrane. This structural role has functional consequences on cAMP and cGMP signaling in a PMCA4-governed microdomain, which ultimately regulates cardiac contractility. In vivo contractility and calcium amplitude were increased in PMCA4 knock-out animals (PMCA4(-/-)) with no change in diastolic relaxation or the rate of calcium decay, showing that PMCA4 has a function distinct from beat-to-beat calcium transport. Surprisingly, in PMCA4(-/-), over 36% of membrane-associated neuronal nitric-oxide synthase (nNOS) protein and activity was delocalized to the cytosol with no change in total nNOS protein, resulting in a significant decrease in microdomain cGMP, which in turn led to a significant elevation in local cAMP levels through a decrease in PDE2 activity (measured by FRET-based sensors). This resulted in increased L-type calcium channel activity and ryanodine receptor phosphorylation and hence increased contractility. In the heart, in addition to subsarcolemmal calcium transport, PMCA4 acts as a structural molecule that maintains the spatial and functional integrity of the nNOS signaling complex in a defined microdomain. This has profound consequences for the regulation of local cyclic nucleotide and hence cardiac β-adrenergic signaling.     

Ubiquitylation of neuronal nitric-oxide synthase by CHIP, a chaperone-dependent E3 ligase

It is established that neuronal nitric-oxide synthase (nNOS) is ubiquitylated and proteasomally degraded. The proteasomal degradation of nNOS is enhanced by suicide inactivation of nNOS or by the inhibition of hsp90, which is a chaperone found in a native complex with nNOS. In the current study, we have examined whether CHIP, a chaperone-dependent E3 ubiquitin-protein isopeptide ligase that is known to ubiquitylate other hsp90-chaperoned proteins, could act as an ubiquitin ligase for nNOS. We found with the use of HEK293T or COS-7 cells and transient transfection methods that CHIP overexpression causes a decrease in immunodetectable levels of nNOS. The extent of the loss of nNOS is dependent on the amount of CHIP cDNA used for transfection. Lactacystin (10 microM), a selective proteasome inhibitor, attenuates the loss of nNOS in part by causing the nNOS to be found in a detergent-insoluble form. Immunoprecipitation of the nNOS and subsequent Western blotting with an anti-ubiquitin IgG shows an increase in nNOS-ubiquitin conjugates because of CHIP. Moreover, incubation of nNOS with a purified system containing an E1 ubiquitin-activating enzyme, an E2 ubiquitin carrier protein conjugating enzyme (UbcH5a), CHIP, glutathione S-transferase-tagged ubiquitin, and an ATP-generating system leads to the ubiquitylation of nNOS. The addition of purified hsp70 and hsp40 to this in vitro system greatly enhances the amount of nNOS-ubiquitin conjugates, suggesting that CHIP is an E3 ligase for nNOS whose action is facilitated by (and possibly requires) its interaction with nNOS-bound hsp70.     

Translocation of endothelial nitric-oxide synthase involves a ternary complex with caveolin-1 and NOSTRIN

Recently, we characterized a novel endothelial nitric-oxide synthase (eNOS)-interacting protein, NOSTRIN (for eNOS-trafficking inducer), which decreases eNOS activity upon overexpression and induces translocation of eNOS away from the plasma membrane. Here, we show that NOSTRIN directly binds to caveolin-1, a well-established inhibitor of eNOS. Because this interaction occurs between the N terminus of caveolin (positions 1-61) and the central domain of NOSTRIN (positions 323-434), it allows for independent binding of each of the two proteins to eNOS. Consistently, we were able to demonstrate the existence of a ternary complex of NOSTRIN, eNOS, and caveolin-1 in Chinese hamster ovary (CHO)-eNOS cells. In human umbilical vein endothelial cells (HUVECs), the ternary complex assembles at the plasma membrane upon confluence or thrombin stimulation. In CHO-eNOS cells, NOSTRIN-mediated translocation of eNOS involves caveolin in a process most likely representing caveolar trafficking. Accordingly, trafficking of NOSTRIN/eNOS/caveolin is affected by altering the state of actin filaments or cholesterol levels in the plasma membrane. During caveolar trafficking, NOSTRIN functions as an adaptor to recruit mediators such as dynamin-2 essential for membrane fission. We propose that a ternary complex between NOSTRIN, caveolin-1, and eNOS mediates translocation of eNOS, with important implications for the activity and availability of eNOS in the cell.     

Interaction of neuronal nitric-oxide synthase with alpha1-syntrophin in rat brain

Neuronal nitric-oxide synthase (nNOS) has a PSD-95/Dlg/ZO-1 (PDZ) domain that can interact with multiple proteins. nNOS has been known to interact with PSD-95 and a related protein, PSD-93, in brain and with alpha1-syntrophin in skeletal muscle in mammals. In this study, we have purified an nNOS-interacting protein from bovine brain using an affinity column made of Sepharose conjugated with glutathione S-transferase-rat nNOS fusion protein and identified it as alpha1-syntrophin by microsequencing. Immunostaining of primary cultures of rat embryonic brain neuronal cells with antibodies against these proteins showed that nNOS and alpha1-syntrophin were colocalized in neuronal cell bodies and neurites. Immunohistochemical analysis indicated that the nNOS- and alpha1-syntrophin-like immunoreactive substances were highly expressed in the rat hypothalamic suprachiasmatic nucleus (SCN) and paraventricular nucleus. In the SCN, nNOS- and alpha1-syntrophin-like immunoreactive substances were colocalized in the same neurons as detected by confocal microscopy. These results indicate that nNOS in brain interacts with alpha1-syntrophin in specific neurons of the SCN and paraventricular nucleus and that this interaction might play a physiological role in functions of these neurons.     

Ubiquitination of neuronal nitric-oxide synthase in vitro and in vivo

It is established that suicide inactivation of neuronal nitric-oxide synthase (nNOS) with guanidine compounds, or inhibition of the hsp90-based chaperone system with geldanamycin, leads to the enhanced proteolytic degradation of nNOS. This regulated proteolysis is mediated, in part, by the proteasome. We show here with the use of human embryonic kidney 293 cells transfected with nNOS that inhibition of the proteasome with lactacystin leads to the accumulation of immunodetectable higher molecular mass forms of nNOS. Some of these higher molecular mass forms were immunoprecipitated by an anti-ubiquitin antibody, indicating that they are nNOS-polyubiquitin conjugates. Moreover, the predominant nNOS-ubiquitin conjugate detected in human embryonic kidney 293 cells, as well as in rat brain cytosol, migrates on SDS-polyacrylamide gels with a mobility near that for the native monomer of nNOS and likely represents a conjugate containing a few or perhaps one ubiquitin. Studies in vitro with the use of (125)I-ubiquitin and reticulocyte extracts could mimic this ubiquitination reaction, which was dependent on ATP. The heme-deficient monomeric form of nNOS is preferentially ubiquitinated over that of the heme-sufficient functionally active homodimer. Thus, we have shown for the first time that ubiquitination of nNOS occurs and is likely involved in the regulated proteolytic removal of non-functional enzyme.     

Guanabenz-mediated inactivation and enhanced proteolytic degradation of neuronal nitric-oxide synthase

Guanabenz, a metabolism-based irreversible inactivator of neuronal nitric-oxide synthase (nNOS) in vitro, causes the loss of immunodetectable nNOS in vivo. This process is selective in that the slowly reversible inhibitor N(G)-nitro-L-arginine did not decrease the levels of nNOS in vivo. To better understand the mechanism for the loss of nNOS protein in vivo, we have investigated the effects of guanabenz and N(G)-nitro-L-arginine in HEK 293 cells stably transfected with the enzyme. We show here that guanabenz, but not N(G)-nitro-L-arginine, caused the inactivation and loss of nNOS protein in the HEK 293 cells. In studies with cycloheximide or in pulse-chase experiments with [(35)S]methionine, we demonstrate that the loss of nNOS was due in large part to enhanced proteolysis of the protein with the half-life decreasing by one-half from 20 to 10 h. Other metabolism-based irreversible inactivators to nNOS, N(G)-methyl-L-arginine, and N(5)-(1-iminoethyl)-L-ornithine, but not the reversible inhibitor 7-nitroindazole (7-NI), caused a similar decrease in the half-life of nNOS. Proteasomal inhibitors, lactacystin, Cbz-leucine-leucine-leucinal, and N-acetyl-leucine-leucine-norleucinal, but not the lysosomal protease inhibitor leupeptin, were found to effectively inhibit the proteolytic degradation of nNOS. Thus we have shown for the first time that the irreversible inactivators of nNOS, perhaps through covalent alteration of the enzyme, enhance the proteolytic turnover of the enzyme by a mechanism involving the proteasome.     

Important role of tetrahydrobiopterin in no complex formation and interdomain electron transfer in neuronal nitric-oxide synthase

Neuronal nitric-oxide synthase (nNOS) is composed of a heme oxygenase domain and a flavin-bound reductase domain. Ca(2+)/calmodulin (CaM) is essential for interdomain electron transfer during catalysis, whereas the role of the catalytically important cofactor, tetrahydrobiopterin (H4B) remains elusive. The product NO appears to bind to the heme and works as a feedback inhibitor. The present study shows that the Fe(3+)-NO complex is reduced to the Fe(2+)-NO complex by NADPH in the presence of both l-Arg and H4B even in the absence of Ca(2+)/CaM. The complex could not be fully reduced in the absence of H4B under any circumstances. However, dihydrobiopterin and N(G)-hydroxy-l-Arg could be substituted for H4B and l-Arg, respectively. No direct correlation could be found between redox potentials of the nNOS heme and the observed reduction of the Fe(3+)-NO complex. Thus, our data indicate the importance of the pterin binding to the active site structure during the reduction of the NO-heme complex by NADPH during catalytic turnover.     

Dystroglycan is not required for localization of dystrophin, syntrophin, and neuronal nitric-oxide synthase at the sarcolemma but regulates integrin alpha 7B expression and caveolin-3 distribution.

Dystroglycan is part of the dystrophin-associated protein complex, which joins laminin in the extracellular matrix to dystrophin within the subsarcolemmal cytoskeleton. We have investigated how mutations in the components of the laminin-dystroglycan-dystrophin axis affect the organization and expression of dystrophin-associated proteins by comparing mice mutant for merosin (alpha(2)-laminin, dy), dystrophin (mdx), and dystroglycan (Dag1) using immunohistochemistry and immunoblots. We report that syntrophin and neuronal nitric-oxide synthase are depleted in muscle fibers lacking both dystrophin and dystroglycan. Some fibers deficient in dystroglycan, however, localize dystrophin at the cell surface at levels similar to that in wild-type muscle. Nevertheless, these fibers have signs of degeneration/regeneration including increased cell surface permeability and central nuclei. In these fibers, syntrophin and nitric-oxide synthase are also localized to the plasma membrane, whereas the sarcoglycan complex is disrupted. These results suggest a mechanism of membrane attachment for dystrophin independent of dystroglycan and that the interaction of sarcoglycans with dystrophin requires dystroglycan. The distribution of caveolin-3, a muscle-specific component of caveolae recently found to bind dystroglycan, was affected in dystroglycan- and dystrophin-deficient mice. We also examined alternative mechanisms of cell-extracellular matrix attachment to elucidate how the muscle basement membrane may subsist in the absence of dystroglycan, and we found the alpha(7B) splice variant of the alpha(7) integrin receptor subunit to be up-regulated. These results support the possibility that alpha(7B) integrin compensates in mediating cell-extracellular matrix attachment but cannot rescue the dystrophic phenotype.     

Distinct influence of N-terminal elements on neuronal nitric-oxide synthase structure and catalysis

Nitric oxide (NO) is a signal molecule produced in animals by three different NO synthases. Of these, only NOS I (neuronal nitric-oxide synthase; nNOS) is expressed as catalytically active N-terminally truncated forms that are missing either an N-terminal leader sequence required for protein-protein interactions or are missing the leader sequence plus three core structural motifs that in other NOS are required for dimer assembly and catalysis. To understand how the N-terminal elements impact nNOS structure-function, we generated, purified, and extensively characterized variants that were missing the N-terminal leader sequence (Delta296nNOS) or missing the leader sequence plus the three core motifs (Delta349nNOS). Eliminating the leader sequence had no impact on nNOS structure or catalysis. In contrast, additional removal of the core elements weakened but did not destroy the dimer interaction, slowed ferric heme reduction and reactivity of a hemedioxy intermediate, and caused a 10-fold poorer affinity toward substrate l-arginine. This created an nNOS variant with slower and less coupled NO synthesis that is predisposed to generate reactive oxygen species along with NO. Our findings help justify the existence of nNOS N-terminal splice variants and identify specific catalytic changes that create functional differences among them.     

Nitric oxide inhibition of ERK1/2 activity in cells expressing neuronal nitric-oxide synthase

Neuronal nitric-oxide synthase (nNOS) is a constitutively expressed enzyme responsible for the production of nitric oxide (NO*) from l-arginine and O2. Nitric oxide is an intra- and intercellular messenger that mediates a diversity of signaling pathways in target cells. In the absence of l-arginine, nNOS has been shown to generate superoxide (O2*). Superoxide, either directly or through its self-dismutation to H2O2, is likewise believed to be a cell-signaling agent. Because nNOS can generate NO* and O2*, we examined the activation of cellular signal transduction pathways in nNOS-transfected cells grown in the presence or absence of l-arginine. Spin trapping/EPR spectroscopy confirmed that stimulated nNOS-transfected cells grown in an l-arginine environment secreted NO* into the surrounding milieu. Production of NO* blocked Ca2+ ionophore-induced activation of the ERK1/2 through a mechanism involving inhibition of the Ras G-protein and Raf-1 kinase. In contrast, ERK activation was largely unaffected in nNOS-transfected cells grown in l-arginine-free media. Inhibition of nNOS-generated NO* with the competitive NOS inhibitor, NG-nitro-l-arginine methyl ester, in cells grown in l-arginine restored ERK1/2 activation to levels similar to that found when nNOS was activated in l-arginine-free media. These findings indicate that nNOS can differentially regulate the ERK signal transduction pathway in a manner dependent on the presence of l-arginine and the production of NO*.     

NIDD, a novel DHHC-containing protein, targets neuronal nitric-oxide synthase (nNOS) to the synaptic membrane through a PDZ-dependent interaction and regulates nNOS activity

Targeting of neuronal nitric-oxide synthase (nNOS) to appropriate sites in a cell is mediated by interactions with its PDZ domain and plays an important role in specifying the sites of reaction of nitric oxide (NO) in the central nervous system. Here we report the identification and characterization of a novel nNOS-interacting DHHC domain-containing protein with dendritic mRNA (NIDD) (GenBank accession number AB098078), which increases nNOS enzyme activity by targeting the nNOS to the synaptic plasma membrane in a PDZ domain-dependent manner. The deduced NIDD protein consisted of 392 amino acid residues and possessed five transmembrane segments, a zinc finger DHHC domain, and a PDZ-binding motif (-EDIV) at its C-terminal tail. In vitro pull-down assays suggested that the C-terminal tail region of NIDD specifically interacted with the PDZ domain of nNOS. The PDZ dependence was confirmed by an experiment using a deletion mutant, and the interaction was further confirmed by co-sedimentation assays using COS-7 cells transfected with NIDD and nNOS. Both NIDD and nNOS were enriched in synaptosome and synaptic plasma membrane fractions and were present in the lipid raft and postsynaptic density fractions in the rat brain. Co-localization of these proteins was also observed by double staining of the proteins in cultured cortical neurons. Thus, NIDD and nNOS were co-localized in the brain, although the colocalizing regions were restricted, as indicated by the distribution of their mRNA expression. Most important, co-transfection of NIDD and nNOS increased NO-producing nNOS activity. These results suggested that NIDD plays an important role in the regulation of the NO signaling pathway at postsynaptic sites through targeting of nNOS to the postsynaptic membrane.     

Differences in a conformational equilibrium distinguish catalysis by the endothelial and neuronal nitric-oxide synthase flavoproteins

Nitric oxide (NO) is a physiological mediator synthesized by NO synthases (NOS). Despite their structural similarity, endothelial NOS (eNOS) has a 6-fold lower NO synthesis activity and 6-16-fold lower cytochrome c reductase activity than neuronal NOS (nNOS), implying significantly different electron transfer capacities. We utilized purified reductase domain constructs of either enzyme (bovine eNOSr and rat nNOSr) to investigate the following three mechanisms that may control their electron transfer: (i) the set point and control of a two-state conformational equilibrium of their FMN subdomains; (ii) the flavin midpoint reduction potentials; and (iii) the kinetics of NOSr-NADP+ interactions. Although eNOSr and nNOSr differed in their NADP(H) interaction and flavin thermodynamics, the differences were minor and unlikely to explain their distinct electron transfer activities. In contrast, calmodulin (CaM)-free eNOSr favored the FMN-shielded (electron-accepting) conformation over the FMN-deshielded (electron-donating) conformation to a much greater extent than did CaM-free nNOSr when the bound FMN cofactor was poised in each of its three possible oxidation states. NADPH binding only stabilized the FMN-shielded conformation of nNOSr, whereas CaM shifted both enzymes toward the FMN-deshielded conformation. Analysis of cytochrome c reduction rates measured within the first catalytic turnover revealed that the rate of conformational change to the FMN-deshielded state differed between eNOSr and nNOSr and was rate-limiting for either CaM-free enzyme. We conclude that the set point and regulation of the FMN conformational equilibrium differ markedly in eNOSr and nNOSr and can explain the lower electron transfer activity of eNOSr.     

Structure and reactivity of a thermostable prokaryotic nitric-oxide synthase that forms a long-lived oxy-heme complex

In an effort to generate more stable reaction intermediates involved in substrate oxidation by nitric-oxide synthases (NOSs), we have cloned, expressed, and characterized a thermostable NOS homolog from the thermophilic bacterium Geobacillus stearothermophilus (gsNOS). As expected, gsNOS forms nitric oxide (NO) from l-arginine via the stable intermediate N-hydroxy l-arginine (NOHA). The addition of oxygen to ferrous gsNOS results in long-lived heme-oxy complexes in the presence (Soret peak 427 nm) and absence (Soret peak 413 nm) of substrates l-arginine and NOHA. The substrate-induced red shift correlates with hydrogen bonding between substrate and heme-bound oxygen resulting in conversion to a ferric heme-superoxy species. In single turnover experiments with NOHA, NO forms only in the presence of H(4)B. The crystal structure of gsNOS at 3.2 AA of resolution reveals great similarity to other known bacterial NOS structures, with the exception of differences in the distal heme pocket, close to the oxygen binding site. In particular, a Lys-356 (Bacillus subtilis NOS) to Arg-365 (gsNOS) substitution alters the conformation of a conserved Asp carboxylate, resulting in movement of an Ile residue toward the heme. Thus, a more constrained heme pocket may slow ligand dissociation and increase the lifetime of heme-bound oxygen to seconds at 4 degrees C. Similarly, the ferric-heme NO complex is also stabilized in gsNOS. The slow kinetics of gsNOS offer promise for studying downstream intermediates involved in substrate oxidation.     

Regioselective nitration of tryptophan by a complex between bacterial nitric-oxide synthase and tryptophanyl-tRNA synthetase

Bacterial nitric-oxide synthase proteins (NOSs) from certain Streptomyces strains have been shown to participate in biosynthetic nitration of tryptophanyl moieties in vivo (Kers, J. A., Wach, M. J., Krasnoff, S. B., Cameron, K. D., Widom, J., Bukhaid, R. A., Gibson, D. M., and Crane, B. R., and Loria, R. (2004) Nature 429, 79-82). We report that the complex between Deinococcus radiodurans NOS (deiNOS) and an unusual tryptophanyl-tRNA synthetase (TrpRS II) catalyzes the regioselective nitration of tryptophan (Trp) at the 4-position. Unlike non-enzymatic Trp nitration, and similar reactions catalyzed by globins and peroxidases, deiNOS only produces the otherwise unfavorable 4-nitro-Trp isomer. Although deiNOS alone will catalyze 4-nitro-Trp production, yields are significantly enhanced by TrpRS II and ATP. 4-Nitro-Trp formation exhibits saturation behavior with Trp (but not tyrosine) and is completely inhibited by the addition of the mammalian NOS cofactor (6R)-5,6,7,8-tetrahydro-l-biopterin (H(4)B). Trp stimulates deiNOS oxidation of substrate l-arginine (Arg) to the same degree as H(4)B. These observations are consistent with a mechanism where Trp or a derivative thereof binds in the NOS pterin site, participates in Arg oxidation, and becomes nitrated at the 4-position.     

Removal of a putative inhibitory element reduces the calcium-dependent calmodulin activation of neuronal nitric-oxide synthase

Neuronal nitric-oxide synthase (NOS) and endothelial NOS are constitutive NOS isoforms that are activated by binding calmodulin in response to elevated intracellular calcium. In contrast, the inducible NOS isoform binds calmodulin at low basal levels of calcium in resting cells. Primary sequence comparisons show that each constitutive NOS isozyme contains a polypeptide segment within its reductase domain, which is absent in the inducible NOS enzyme. To study a possible link between the presence of these additional polypeptide segments in constitutive NOS enzymes and their calcium-dependent calmodulin activation, three deletion mutants were created. The putative inhibitory insert was removed from the FMN binding regions of the neuronal NOS holoenzyme and from two truncated neuronal NOS reductase enzymes in which the calmodulin binding region was either included or deleted. All three mutant enzymes showed reduced incorporation of FMN and required reconstitution with exogenous FMN for activity. The combined removal of both the calmodulin binding domain and the putative inhibitory insert did not result in a calmodulin-independent neuronal NOS reductase. Thus, although the putative inhibitory element has an effect on the calcium-dependent calmodulin activation of neuronal NOS, it does not have the properties of the typical autoinhibitory domain found in calmodulin-activated enzymes.