Electron paramagnetic resonance characterization of tetrahydrobiopterin radical formation in bacterial nitric oxide synthase compared to mammalian nitric oxide synthase

H(4)B is an essential catalytic cofactor of the mNOSs. It acts as an electron donor and activates the ferrous heme-oxygen complex intermediate during Arg oxidation (first step) and NOHA oxidation (second step) leading to nitric oxide and citrulline as final products. However, its role as a proton donor is still debated. Furthermore, its exact involvement has never been explored for other NOSs such as NOS-like proteins from bacteria. This article proposes a comparative study of the role of H(4)B between iNOS and bsNOS. In this work, we have used freeze-quench to stop the arginine and NOHA oxidation reactions and trap reaction intermediates. We have characterized these intermediates using multifrequency electron paramagnetic resonance. For the first time, to our knowledge, we report a radical formation for a nonmammalian NOS. The results indicate that bsNOS, like iNOS, has the capacity to generate a pterin radical during Arg oxidation. Our current electron paramagnetic resonance data suggest that this radical is protonated indicating that H(4)B may not transfer any proton. In the 2nd step, the radical trapped for iNOS is also suggested to be protonated as in the 1st step, whereas it was not possible to trap a radical for the bsNOS 2nd step. Our data highlight potential differences for the catalytic mechanism of NOHA oxidation between mammalian and bacterial NOSs.     

CO binding studies of nitric oxide synthase: effects of the substrate, inhibitors and tetrahydrobiopterin

The dissociation constant (K_d) for CO from neuronal nitric oxide synthase heme in the absence of the substrate and cofactor was less than 10⁻³ μM. In the presence of

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-Arg, it dramatically increased up to 1 μM. In the presence of inhibitors such as N^G-nitro-

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-arginine methyl ester and 7-nitroindazole (NI), the K_d value further increased up to more than 100 μM. Addition of the cofactor, 5,6,7,8-tetrahydrobiopterin (H₄B), increased the K_d value by 10-fold in the presence of

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-Arg, whereas it decreased the value to less than one 250th in the presence of NI. Addition of H₄B increased the recombination rate constant (k_on) for CO by more than two-fold in the presence of

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-Arg or N⁶-(1-iminoethyl)-

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-lysine, whereas it decreased the k_on value by three-fold in the presence of

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-thiocitrulline. Thus, the binding fashion of some of inhibitors, such as NI, may be different from that of

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-Arg with respect to the H₄B effect.     

Macrophage nitric oxide synthase: relationship between enzyme-bound tetrahydrobiopterin and synthase activity.

Nitric oxide synthase (NOS) (EC 1.14.23) catalyzes the oxidation of L-arginine to citrulline and nitric oxide. The complex reaction carried out by NOS, which involves NADPH, O2, and enzyme-bound FAD, FMN, and tetrahydrobiopterin (BH4), has only recently begun to be elucidated. Herein we report the characterization of the pterin requirement of murine macrophage NOS. Although purified NOS activity was not dependent on BH4, activity was significantly enhanced by BH4 in a concentration-dependent fashion. NOS purified in the absence of added BH4 was found to contain substoichiometric concentrations of enzyme-bound pterin, where increased concentrations of bound pterin correlated with an increase in activity when assayed in the absence of exogenous BH4. However, NOS purified in the presence of BH4 followed by gel filtration exhibited a 1 mol of pterin:1 mol of NOS 130-kDa subunit stoichiometry and activity that was essentially independent of exogenous BH4. Experiments to probe a redox role for the pterin were carried out using pterin analogues. 6(R,S)-Methyltetrahydropterin was found to increase NOS activity in enzyme purified in the absence of BH4. However, the deaza analogue, 6(R,S)-methyl-5-deazatetrahydropterin, was not only incapable of supporting enzymatic turnover but also inhibited citrulline formation in a concentration-dependent manner. Overall, these results support a role for BH4 in the NOS reaction that involves stabilization of the enzyme and redox chemistry wherein a 1:1 stoichiometry between bound pterin and NOS subunit results in maximum activity.     

Nitric oxide and nitric oxide synthase activity in plants

Research on NO in plants has gained considerable attention in recent years mainly due to its function in plant growth and development and as a key signalling molecule in different intracellular processes in plants. The NO emission from plants is known since the 1970s, and now there is abundant information on the multiple effects of exogenously applied NO on different physiological and biochemical processes of plants. The physiological function of NO in plants mainly involves the induction of different processes, including the expression of defence-related genes against pathogens and apoptosis/programmed cell death (PCD), maturation and senescence, stomatal closure, seed germination, root development and the induction of ethylene emission. NO can be produced in plants by non-enzymatic and enzymatic systems. The NO-producing enzymes identified in plants are nitrate reductase, and several nitric oxide synthase-like activities, including one localized in peroxisomes which has been biochemically characterized. Recently, two genes of plant proteins with NOS activity have been isolated and characterized for the first time, and both proteins do not have sequence similarities to any mammalian NOS isoform. However, different evidence available indicate that there are other potential enzymatic sources of NO in plants, including xanthine oxidoreductase, peroxidase, cytochrome P450, and some hemeproteins. In plants, the enzymatic production of the signal molecule NO, either constitutive or induced by different biotic/abiotic stresses, may be a much more common event than was initially thought.     

Structures of tetrahydrobiopterin binding-site mutants of inducible nitric oxide synthase oxygenase dimer and implicated roles of Trp457.

To better understand potential roles of conserved Trp457 of the murine inducible nitric oxide synthase oxygenase domain (iNOS(ox); residues 1-498) in maintaining the structural integrity of the (6R)-5,6,7,8-tetrahydrobiopterin (H(4)B) binding site located at the dimer interface and in supporting H(4)B redox activity, we determined crystallographic structures of W457F and W457A mutant iNOS(ox) dimers (residues 66-498). In W457F iNOS(ox), all the important hydrogen-bonding and aromatic stacking interactions that constitute the H(4)B binding site and that bridge the H(4)B and heme sites are preserved. In contrast, the W457A mutation results in rearrangement of the Arg193 side chain, orienting its terminal guanidinium group almost perpendicular to the ring plane of H(4)B. Although Trp457 is not required for dimerization, both Trp457 mutations led to the increased mobility of the N-terminal H(4)B binding segment (Ser112-Met114), which might indicate reduced stability of the Trp457 mutant dimers. The Trp457 mutant structures show decreased pi-stacking with bound pterin when the wild-type pi-stacking Trp457 position is occupied with the smaller Phe457 in W457F or positive Arg193 in W457A. The reduced pterin pi-stacking in these mutant structures, relative to that in the wild-type, implies stabilization of reduced H(4)B and destabilization of the pterin radical, consequently slowing electron transfer to the heme ferrous-dioxy (Fe(II)O(2)) species during catalysis. These crystal structures therefore aid elucidation of the roles and importance of conserved Trp457 in maintaining the structural integrity of the H(4)B binding site and of H(4)B-bound dimers, and in influencing the rate of electron transfer between H(4)B and heme in NOS catalysis.     

Nitric oxide metabolism in mammalian cells: substrate and inhibitor profiles of a NADPH-cytochrome P450 oxidoreductase-coupled microsomal nitric oxide dioxygenase

Human intestinal Caco-2 cells metabolize and detoxify NO via a dioxygen- and NADPH-dependent, cyanide- and CO-sensitive pathway that yields nitrate. Enzymes catalyzing NO dioxygenation fractionate with membranes and are enriched in microsomes. Microsomal NO metabolism shows apparent KM values for NO, O2, and NADPH of 0.3, 9, and 2 microM, respectively, values similar to those determined for intact or digitonin-permeabilized cells. Similar to cellular NO metabolism, microsomal NO metabolism is superoxide-independent and sensitive to heme-enzyme inhibitors including CO, cyanide, imidazoles, quercetin, and allicin-enriched garlic extract. Selective inhibitors of several cytochrome P450s and heme oxygenase fail to inhibit the activity, indicating limited roles for a subset of microsomal heme enzymes in NO metabolism. Diphenyleneiodonium and cytochrome c(III) inhibit NO metabolism, suggesting a role for the NADPH-cytochrome P450 oxidoreductase (CYPOR). Involvement of CYPOR is demonstrated by the specific inhibition of the NO metabolic activity by inhibitory anti-CYPOR IgG. In toto, the results suggest roles for a microsomal CYPOR-coupled and heme-dependent NO dioxygenase in NO metabolism, detoxification, and signal attenuation in mammalian cells.     

Structure of tetrahydrobiopterin tunes its electron transfer to the heme-dioxy intermediate in nitric oxide synthase

How 6R-tetrahydrobiopterin (H(4)B) participates in Arg hydroxylation as catalyzed by the nitric oxide synthases (NOSs) is a topic of current interest. Previous work with the oxygenase domain of inducible NOS (iNOSoxy) demonstrated that H(4)B radical formation is kinetically coupled to disappearance of an initial heme-dioxy intermediate and to Arg hydroxylation in a single turnover reaction run at 10 degrees C [Wei, C.-C., Wang, Z.-Q., Wang, Q., Meade, A. L., Hemann, C., Hille, R., and Stuehr, D. J. (2001) J. Biol. Chem. 276, 315-319]. Here we used 5-methyl-H(4)B to investigate how pterin structure influences radical formation and associated catalytic steps. In the presence of Arg, the heme-dioxy intermediate in 5-methyl-H(4)B-bound iNOSoxy reacted at a rate of 35 s(-)(1), which is 3-fold faster than with H(4)B. This was coupled to a faster rate of 5-methyl-H(4)B radical formation (40 vs 12.5 s(-)(1)) and to a faster and more productive Arg hydroxylation. The EPR spectrum of the enzyme-bound 5-methyl-H(4)B radical had different hyperfine structure than the bound H(4)B radical and exhibited a 3-fold longer half-life after its formation. A crystal structure of 5-methyl-H(4)B-bound iNOSoxy revealed that there are minimal changes in conformation of the bound pterin or in its interactions with the protein as compared to H(4)B. Together, we conclude the following: (1) The rate of heme-dioxy reduction is linked to pterin radical formation and is sensitive to pterin structure. (2) Faster heme-dioxy reduction increases the efficiency of Arg hydroxylation but still remains rate limiting for the reaction. (3) The 5-methyl group influences heme-dioxy reduction by altering the electronic properties of the pterin rather than changing protein structure or interactions. (4) Faster electron transfer from 5-methyl-H(4)B may be due to increased radical stability afforded by the N-5 methyl group.     

Stimulation of human nitric oxide synthase by tetrahydrobiopterin and selective binding of the cofactor.

To check the stimulatory potency of the tetrahydro forms of the two major pteridines occurring in human tissues, neopterin and biopterin, NO synthase was purified 6000-fold from human cerebellum. Tetrahydrobiopterin stimulated the activity up to 4.5-fold in a concentration dependent manner with a maximum above 1 microM, whereas tetrahydroneopterin was completely inactive in concentrations up to 100 microM. Tetrahydrobiopterin, but not neopterin derivatives, were copurified with the NO synthase activity. Our results demonstrate that human cerebellum contains a tetrahydrobiopterin dependent NO synthase activity.     

Nitric oxide synthase like activity-dependent nitric oxide production protects against chilling-induced oxidative damage in Chorispora bungeana suspension cultured cells

In the present study, we used suspension cultured cells from Chorispora bungeana Fisch. and C.A. Mey to investigate whether nitric oxide (NO) is involved in the signaling pathway of chilling adaptive responses. Low temperatures at 4 °C or 0 °C induced ion leakage, lipid peroxidation and cell viability suppression, which were dramatically alleviated by exogenous application of NO donor sodium nitroprusside (SNP). The levels of reactive oxygen species (ROS) were obviously reduced, and the activities of antioxidant enzymes such as ascorbate peroxidase (APX, EC 1.11.1.11), catalase (CAT, EC 1.11.1.6), glutathione reductase (GR, EC 1.6.4.2), peroxidase (POD, EC 1.11.1.7) and superoxide dismutase (SOD, EC 1.15.1.1) and the contents of ascorbic acid (AsA) and reduced glutathione (GSH) increased evidently in the presence of SNP under chilling stress. In addition, under low temperature conditions, treatment with NO scavenger PTIO or mammalian NO synthase (NOS) inhibitor l-NAME remarkably aggravated oxidative damage in the suspension cultures compared with that of chilling treatment alone. Moreover, measurements of NOS activity and NO production showed that both NOS activity and endogenous NO content increased markedly under chilling stress. The accumulation of NO was inhibited by l-NAME in chilling-treated cultures, indicating that most NO production under chilling may be generated from NOS-like activity. Collectively, these results suggest that chilling-induced NO accumulation can effectively protect against oxidative injury and that NOS like activity-dependent NO production might act as an antioxidant directly scavengering ROS or operate as a signal activating antioxidant defense under chilling stress, thus conferring an increased tolerance to chilling in C. bungeana suspension cultures.     

Nitric oxide and nitric oxide synthase isoforms in the normal, hypertrophic, and failing heart

Increased oxidative stress is one of the basic contributors to the development of the cardiovascular complications in diabetes. Both endothelial and vascular smooth muscle cell dysfunctions are the main sign involved in the pathogenesis of diabetic cardiovascular dysfunction. Matrix metalloproteinases (MMPs) are expressed in the vasculature, and participate in tissue remodeling under pathological conditions such as increased oxidative stress, whereas little is known about effect of hyperglycemia on regulation of MMPs in vascular system. Therefore, we aimed to evaluate the effect of an antioxidant, sodium selenate treatment (0.3 mg/kg for 4 weeks) on function of streptozotocin-diabetic rat aorta. Sodium selenate treatment improved significantly impaired isoproterenol-induced relaxation responses and contraction responses of the aortic strips, and exhibited marked protection against diabetes-induced degenerative changes in the smooth muscle cell morphology. Biochemical data showed that sodium selenate treatment induced a significant regulation of MMP-2 activity and protein loss as well as normalization of increased levels of tissue nitrite and protein thiol oxidation. In addition, this treatment restored diabetes-induced increased levels of endothelin-1, PKC, and cAMP production in the aortic tissue. Taken together, our data demonstrate that these beneficial effects of sodium selenate treatment in diabetics are related to be not only inhibition of increased oxidative stress but also prevention of both receptor- and smooth muscle-mediated dysfunction of vasculature, in part, via regulation of MMP-2. Such an observation provides evidence for potential therapeutic usage of selenium compounds for the amelioration of vascular disorders in diabetes.     

Inducible nitric oxide synthase: role of the N-terminal beta-hairpin hook and pterin-binding segment in dimerization and tetrahydrobiopterin interaction

The oxygenase domain of the inducible nitric oxide synthase (iNOSox; residues 1-498) is a dimer that binds heme, L-arginine and tetrahydrobiopterin (H(4)B) and is the site for nitric oxide synthesis. We examined an N-terminal segment that contains a beta-hairpin hook, a zinc ligation center and part of the H(4)B-binding site for its role in dimerization, catalysis, and H(4)B and substrate interactions. Deletion mutagenesis identified the minimum catalytic core and indicated that an intact N-terminal beta-hairpin hook is essential. Alanine screening mutagenesis of conserved residues in the hook revealed five positions (K82, N83, D92, T93 and H95) where native properties were perturbed. Mutants fell into two classes: (i) incorrigible mutants that disrupt side-chain hydrogen bonds and packing interactions with the iNOSox C-terminus (N83, D92 and H95) and cause permanent defects in homodimer formation, H(4)B binding and activity; and (ii) reformable mutants that destabilize interactions of the residue main chain (K82 and T93) with the C-terminus and cause similar defects that were reversible with high concentrations of H(4)B. Heterodimers comprised of a hook-defective iNOSox mutant subunit and a full-length iNOS subunit were active in almost all cases. This suggests a mechanism whereby N-terminal hooks exchange between subunits in solution to stabilize the dimer.     

Nitric oxide synthase immunoreactivity in the nematode Trichinella britovi. Evidence for nitric oxide production by the parasite

Nitric oxide has been extensively studied as an effector molecule of the host immune response against both protozoa and helminths, but parasites can also produce this molecule, through the action of nitric oxide (NO) synthases or NO synthases-like enzymes. The aim of this study was to verify the possible production of NO by Trichinella britovi L(1) larvae and the enzymes involved in this process. The NO synthase immunoreactivity and putative nitric oxide synthase-activity was analysed using antibodies to mammalian NO synthase III and to nitrotyrosine with immunohistochemistry, gold immunocytochemistry and immunoblot analysis and NADPH-diaphorase histochemistry. Our results show that T. britovi L(1) larvae possess an enzymatic activity capable of producing NO. The localisation of this activity, according to the NADPH-diaphorase histochemistry, is both at the cuticular and the internal level. This localisation is confirmed by nitrotyrosine immunohistochemistry both under optical and electron microscopy. Using the NO synthase III antibody, a similar pattern of labelling was found: in particular, electron microscopy showed a localisation of this immunoreactivity in the cuticle and in the stichocytes, where only the alpha2 granules contained gold particles, mainly concentrated at their periphery. Four polypeptides reacting to the NO synthase III antibody are revealed by Western blotting. Their molecular weight ranged from 38 to 50 kDa. A significant reaction of the anti-nitrotyrosine antibody to polypeptides 95, 60, 48 and 39 kDa from the same sample suggested the presence of different nitrosylated proteins.     

Reactions catalyzed by the heme domain of inducible nitric oxide synthase: evidence for the involvement of tetrahydrobiopterin in electron transfer

The heme domain (iNOS(heme)) of inducible nitric oxide synthase (iNOS) was expressed in Escherichia coli and purified to homogeneity. Characterization of the expressed iNOS(heme) shows it to behave in all respects like full-length iNOS. iNOS(heme) is isolated without bound pterin but can be readily reconstituted with (6R)-5,6,7,8-tetrahydro-L-biopterin (H(4)B) or other pterins. The reactivity of pterin-bound and pterin-free iNOS(heme) was examined, using sodium dithionite as the reductant. H(4)B-bound iNOS(heme) catalyzes both steps of the NOS reaction, hydroxylating arginine to N(G)-hydroxy-L-arginine (NHA) and oxidizing NHA to citrulline and *NO. Maximal product formation (0.93 plus minus 0.12 equiv of NHA from arginine and 0.83 plus minus 0.08 equiv of citrulline from NHA) requires the addition of 2 to 2.5 electron equiv. Full reduction of H(4)B-bound iNOS(heme) with dithionite also requires 2 to 2.5 electron equiv. These data together demonstrate that fully reduced H(4)B-bound iNOS(heme) is able to catalyze the formation of 1 equiv of product in the absence of electrons from dithionite. Arginine hydroxylation requires the presence of a bound, redox-active tetrahydropterin; pterin-free iNOS(heme) or iNOS(heme) reconstituted with a redox-inactive analogue, 6(R,S)-methyl-5-deaza-5,6,7,8-tetrahydropterin, did not form NHA under these conditions. H(4)B has an integral role in NHA oxidation as well. Pterin-free iNOS(heme) oxidizes NHA to citrulline, N(delta)-cyanoornithine, an unidentified amino acid, and NO(-). Maximal product formation (0.75 plus minus 0.01 equiv of amino acid products) requires the addition of 2 to 2.5 electron equiv, but reduction of pterin-free iNOS(heme) requires only 1 to 1.5 electron equiv, indicating that both electrons for the oxidation of NHA by pterin-free iNOS(heme) are derived from dithionite. These data provide strong evidence that H(4)B is involved in electron transfer in NOS catalysis.     

Differing roles of mitochondrial nitric oxide synthase in cardiomyocytes and urothelial cells

The existence of mitochondrial nitric oxide (NO) synthase (mtNOS) has been controversial since it was first reported in 1995. We have addressed this issue by making direct microsensor measurements of NO production in the mitochondria isolated from mouse hearts. Mitochondrial NO production was stimulated by Ca2+ and inhibited by blocking electrogenic Ca2+ uptake or by using NOS antagonists. Cardiac mtNOS was identified as the neuronal isoform by the absence of NO production in the mitochondria of mice lacking the neuronal but not the endothelial or inducible isoforms. In cardiomyocytes from dystrophin-deficient (mdx) mice, elevated intracellular Ca2+, increased mitochondrial NO production, slower oxidative phosphorylation, and decreased ATP production were detected. Inhibition of mtNOS increased contractility in mdx but not in wild-type cardiomyocytes, indicating that mtNOS may protect the cells from overcontracting. mtNOS was also implicated in radiation-induced cell damage. In irradiated rat/mouse urinary bladders, we have evidence that mitochondrially produced NO damages the urothelial "umbrella" cells that line the bladder lumen. This damage disrupts the permeability barrier thereby creating the potential to develop radiation cystitis. RT-PCR and Southern blot analyses indicate that mtNOS is restricted to the umbrella cells, which scanning electron micrographs show are selectively damaged by radiation. Simultaneous microsensor measurements demonstrate that radiation increases NO and peroxynitrite (ONOO-) production in these cells, which can be prevented by transfection with manganese superoxide dismutase (MnSOD) or instillation of NOS antagonists during irradiation or irradiation of bladders devoid of mtNOS. These studies demonstrate that mtNOS is in the cardiomyocytes and urothelial cells, that it is derived from the neuronal isoform, and that it can be either protective or detrimental.     

Nitric oxide in skeletal muscle: inhibition of nitric oxide synthase inhibits walking speed in rats.

Nitric oxide (NO*) is a multifunctional messenger molecule generated by a family of enzymes called the nitric oxide synthases (NOSs). Although NOSs have been identified in skeletal muscle, specifically brain NOS (bNOS) and endothelial NOS (eNOS), their role has not been well clarified. The goals of this investigation were to (1) characterize the immunoreactivity, Ca(2+) dependence, and activity of NOS in human and rat skeletal muscle and (2) using a rat model, investigate the effect of chronic blockade of NOS on skeletal muscle structure and function. Our results showed that both human and rodent skeletal muscle had NOS activity. This NOS activity was similar to that of the endothelial and brain NOS isoforms in that it was calcium-dependent. However, Western blot analysis consistently showed that a polyclonal antibody raised against a peptide sequence of human inducible NOS (iNOS) reacted with a protein with a molecular weight (95 kDa) that was different from that of other NOS isoforms. RT-PCR analysis identified the mRNA expression of not only eNOS and bNOS but also iNOS in human and rat muscle. Inhibition of nitric oxide synthase in rats with N(omega)-nitro-L-arginine methyl ester (L-NAME) resulted in a progressive, severe reduction in walking speed (30-fold reduction in walking velocity at day 22, P < 0.001), muscle fiber cross-sectional area (40% reduction at day 22, P < 0.001), and muscle mass (40% reduction in dry weight at day 22, P < 0.01). Rats fed the same regimen of the enantiomer of L-NAME (d-NAME) had normal motor function, muscle fiber morphology, and muscle mass. Taken together, these results imply that there may be a novel nitric oxide synthase in muscle and that NO. generated from muscle may be important in muscle function.     

Redox function of tetrahydrobiopterin and effect of L-arginine on oxygen binding in endothelial nitric oxide synthase

Tetrahydrobiopterin (BH(4)), not dihydrobiopterin or biopterin, is a critical element required for NO formation by nitric oxide synthase (NOS). To elucidate how BH(4) affects eNOS activity, we have investigated BH(4) redox functions in the endothelial NOS (eNOS). Redox-state changes of BH(4) in eNOS were examined by chemical quench/HPLC analysis during the autoinactivation of eNOS using oxyhemoglobin oxidation assay for NO formation at room temperature. Loss of NO formation activity linearly correlated with BH(4) oxidation, and was recovered by overnight incubation with fresh BH(4). Thus, thiol reagents commonly added to NOS enzyme preparations, such as dithiothreitol and beta-mercaptoethanol, probably preserve enzyme activity by preventing BH(4) oxidation. It has been shown that conversion of L-arginine to N-hydroxy-L-arginine in the first step of NOS catalysis requires two reducing equivalents. The first electron that reduces ferric to the ferrous heme is derived from flavin oxidation. The issue of whether BH(4) supplies the second reducing equivalent in the monooxygenation of eNOS was investigated by rapid-scan stopped-flow and rapid-freeze-quench EPR kinetic measurements. In the presence of L-arginine, oxygen binding kinetics to ferrous eNOS or to the ferrous eNOS oxygenase domain (eNOS(ox)) followed a sequential mechanism: Fe(II) <--> Fe(II)O(2) --> Fe(III) + O(2)(-). Without L-arginine, little accumulation of the Fe(II)O(2) intermediate occurred and essentially a direct optical transition from the Fe(II) form to the Fe(III) form was observed. Stabilization of the Fe(II)O(2) intermediate by L-arginine has been established convincingly. On the other hand, BH(4) did not have significant effects on the oxygen binding and decay of the oxyferrous intermediate of the eNOS or eNOS oxygenase domain. Rapid-freeze-quench EPR kinetic measurements in the presence of L-arginine showed a direct correlation between BH(4) radical formation and decay of the Fe(II)O(2) intermediate, indicating that BH(4) indeed supplies the second electron for L-arginine monooxygenation in eNOS.     

Nitric oxide synthase in filariae: demonstration of nitric oxide production by embryos in Brugia malayi and Acanthocheilonema viteae

The radical gas nitric oxide (NO) is synthesized by nitric oxide synthase (NOS) from l-arginine and molecular oxygen. Nitric oxide is an important signaling molecule in invertebrate and vertebrate systems. Previously we have shown that NOS is localized to more tissues in Brugia malayi than has been reported in Ascaris suum. In this paper, we analyze the distribution of NOS in Acanthocheilonema viteae, a filarial nematode that differs from B. malayi in that A. viteae females release microfilariae without a sheath. A. viteae is also one of a few filarial parasites without the Wolbachia intracellular endosymbiont. By use of a specific antibody, NOS was demonstrated in extracts of A. viteae and Dirofilaria immitis. The localization pattern of NOS in A. viteae was similar to that seen in B. malayi, with the enzyme localized to the body wall muscles of both sexes, developing spermatozoa, intrauterine sperm, and early embryos. By use of DAF-2, a fluorescent indicator specific for nitric oxide, the embryos of B. malayi and A. viteae were demonstrated to produce NO ex utero. The near identical staining patterns seen in A. viteae and B. malayi argue that NO is not produced by Wolbachia, nor is it produced by the nematodes in response to the infection. Localization of NOS to the sperm of filarial nematodes suggests a role for NO during fertilization as has been described for sea urchin and ascidian fertilization. Demonstration of the activity of embryonic NOS supports our earlier hypothesis that NO is a signaling molecule during embryogenesis in filarial nematodes.     

Low-temperature optical absorption spectra suggest a redox role for tetrahydrobiopterin in both steps of nitric oxide synthase catalysis

To investigate the role of tetrahydrobiopterin (BH4) in the catalytic mechanism of nitric oxide synthase (NOS), we analyzed the spectral changes following addition of oxygen to the reduced oxygenase domain of endothelial nitric oxide synthase (NOS) in the presence of different pteridines at -30 degrees C. In the presence of N(G)-hydroxy-L-arginine (NOHLA) and BH4 or 5-methyl-BH4, both of which support NO synthesis, the first observable species were mixtures of high-spin ferric NOS (395 nm), ferric NO-heme (439 nm), and the oxyferrous complex (417 nm). With Arg, no clear intermediates could be observed under the same conditions. In the presence of the BH4-competitive inhibitor 7,8-dihydrobiopterin (BH2), intermediates with maxima at 417 and 425 nm were formed in the presence of Arg and NOHLA, respectively. In the presence of 4-amino-BH4, the maxima of the intermediates with Arg and NOHLA were at 431 and 423 nm, respectively. We ascribe all four spectra to oxyferrous heme complexes. The intermediates observed in this study slowly decayed to the high-spin ferric state at -30 degrees C, except for those formed in the presence of 4-amino-BH4, which required warming to room temperature for regeneration of high-spin ferric NOS; with Arg, regeneration remained incomplete. From these observations, we draw several conclusions. (1) BH4 is required for reductive oxygen activation, probably as a transient one-electron donor, not only in the reaction with Arg but also with NOHLA; (2) in the absence of redox-active pterins, reductive oxygen activation does not occur, which results in accumulation of the oxyferrous complex; (3) the spectral properties of the oxyferrous complex are affected by the presence and identity of the substrate; (4) the slow and incomplete formation of high-spin ferric heme with 4-amino-BH4 suggests a structural cause for inhibition of NOS activity by this pteridine.