Mapping the Intersubunit Interdomain FMN-Heme Interactions in Neuronal Nitric Oxide Synthase by Targeted Quantitative Cross-Linking Mass Spectrometry.

Nitric oxide synthase (NOS) in mammals is a family of multidomain proteins in which interdomain electron transfer (IET) is controlled by domain-domain interactions. Calmodulin (CaM) binds to the canonical CaM-binding site in the linker region between the FMN and heme domains of NOS and allows tethered FMN domain motions, enabling an intersubunit FMN-heme IET in the output state for NO production. Our previous cross-linking mass spectrometric (XL MS) results demonstrated site-specific protein dynamics in the CaM-responsive regions of rat neuronal NOS (nNOS) reductase construct, a monomeric protein [Jiang et al., Biochemistry, 2023, 62, 2232-2237]. In this work, we have extended our combined approach of XL MS structural mapping and AlphaFold structural prediction to examine the homodimeric nNOS oxygenase/FMN (oxyFMN) construct, an established model of the NOS output state. We employed parallel reaction monitoring (PRM) based quantitative XL MS (qXL MS) to assess the CaM-induced changes in interdomain dynamics and interactions. Intersubunit cross-links were identified by mapping the cross-links onto top AlphaFold structural models, which was complemented by comparing their relative abundances in the cross-linked dimeric and monomeric bands. Furthermore, contrasting the CaM-free and CaM-bound nNOS samples shows that CaM enables the formation of the intersubunit FMN-heme docking complex and that CaM binding induces extensive, allosteric conformational changes across the NOS regions. Moreover, the observed cross-links sites specifically respond to changes in ionic strength. This indicates that interdomain salt bridges are responsible for stabilizing and orienting the output state for efficient FMN-heme IET. Taken together, our targeted qXL MS results have revealed that CaM and ionic strength modulate specific dynamic changes in the CaM/FMN/heme complexes, particularly in the context of intersubunit interdomain FMN-heme interactions.

2-Aminopyridines with a shortened amino sidechain as potent, selective, and highly permeable human neuronal nitric oxide synthase inhibitors.

A series of potent, selective, and highly permeable human neuronal nitric oxide synthase inhibitors (hnNOS) based on the 2-aminopyridine scaffold with a shortened amino sidechain is reported. A rapid and simple protocol was developed to access these inhibitors in excellent yields. Neuronal nitric oxide synthase (nNOS) is a novel therapeutic target for the treatment of various neurological disorders. The major challenges in designing nNOS inhibitors in humans focus on potency, selectivity over other isoforms of nitric oxide synthases (NOSs), and blood-brain barrier permeability. In this context, we discovered a promising inhibitor, 6-(3-(4,4-difluoropiperidin-1-yl)propyl)-4-methylpyridin-2-amine dihydrochloride, that exhibits excellent potency for rat (Ki = 46 nM) and human nNOS (Ki = 48 nM), respectively, with 388-fold human eNOS and 135-fold human iNOS selectivity. It also displayed excellent permeability (Pe = 17.3 × 10-6 cm s-1) through a parallel artificial membrane permeability assay, a model for blood-brain permeability. We found that increasing lipophilicity by incorporation of fluorine atoms on the backbone of the inhibitors significantly increased potential blood-brain barrier permeability. In addition to measuring potency, isoform selectivity, and permeability of NOS inhibitors, we also explored structure-activity relationships via structures of key inhibitors complexed to various isoforms of nitric oxide synthases.

Heat shock protein 90α increases superoxide generation from neuronal nitric oxide synthases.

Neuronal nitric oxide synthase (nNOS) generates superoxide, particularly at sub-optimal l-arginine (l-Arg) substrate concentrations. Heat shock protein 90 (Hsp90) was reported to inhibit superoxide generation from nNOS protein. However, commercially available Hsp90 product from bovine brain tissues with unspecified Hsp90α and Hsp90ß contents and an undefined Hsp90 protein oligomeric state was utilized. These two Hsp90s can have opposite effect on superoxide production by NOS. Importantly, emerging evidence indicates that nNOS splice variants are involved in different biological functions by functioning distinctly in redox signaling. In the present work, purified recombinant human Hsp90α, in its native dimeric state, was used in electron paramagnetic resonance (EPR) spin trapping experiments to study the effects of Hsp90α on superoxide generation from nNOS splice variants nNOSµ and nNOSα. Human Hsp90α was found to significantly increase superoxide generation from nNOSµ and nNOSα proteins under l-Arg-depleted conditions and Hsp90α influenced superoxide production by nNOSµ and nNOSα at varying degrees. Imidazole suppressed the spin adduct signal, indicating that superoxide was produced at the heme site of nNOS in the presence of Hsp90α, whereas l-Arg repletion diminished superoxide production by the nNOS-Hsp90α. Moreover, NADPH consumption rate values exhibited a similar trend/difference as a function of Hsp90α and l-Arg. Together, these EPR spin trapping and NADPH oxidation kinetics results demonstrated noticeable Hsp90α-induced increases in superoxide production by nNOS and a distinguishable effect of Hsp90α on nNOSµ and nNOSα proteins.

Coordination between Calcium/Calmodulin-Dependent Protein Kinase II and Neuronal Nitric Oxide Synthase in Neurons.

Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) is highly abundant in the brain and exhibits broad substrate specificity, thereby it is thought to participate in the regulation of neuronal death and survival. Nitric oxide (NO), produced by neuronal NO synthase (nNOS), is an important neurotransmitter and plays a role in neuronal activity including learning and memory processes. However, high levels of NO can contribute to excitotoxicity following a stroke and neurodegenerative disease. Aside from NO, nNOS also generates superoxide which is involved in both cell injury and signaling. CaMKII is known to activate and translocate from the cytoplasm to the post-synaptic density in response to neuronal activation where nNOS is predominantly located. Phosphorylation of nNOS at Ser847 by CaMKII decreases NO generation and increases superoxide generation. Conversely, NO-induced S-nitrosylation of CaMKII at Cys6 is a prominent determinant of the CaMKII inhibition in ATP competitive fashion. Thus, the "cross-talk" between CaMKII and NO/superoxide may represent important signal transduction pathways in brain. In this review, we introduce the molecular mechanism of and pathophysiological role of mutual regulation between CaMKII and nNOS in neurons.

Heat shock protein 90 enhances the electron transfer between the FMN and heme cofactors in neuronal nitric oxide synthase.

Heat shock protein 90 (Hsp90) is a key regulator of nitric oxide synthase (NOS) in vivo. Despite its functional importance, little is known about the underlying molecular mechanism. Here, purified dimeric human Hsp90α was used to investigate whether (and if so, how) Hsp90 affects the FMN-heme interdomain electron transfer (IET) step in NOS. Hsp90α increases the IET rate for rat neuronal NOS (nNOS) in a dose-saturable manner, and a single charge-neutralization mutation at conserved Hsp90 K585 abolishes the effect. The kinetic results with added Ficoll 70, a crowder, further indicate that Hsp90 enhances the FMN-heme IET through specific association with nNOS. The Hsp90-nNOS docking models provide hints on the putative role of Hsp90 in constraining the available conformational space for the FMN domain motions.

First Contact: 7-Phenyl-2-Aminoquinolines, Potent and Selective Neuronal Nitric Oxide Synthase Inhibitors That Target an Isoform-Specific Aspartate.

Inhibition of neuronal nitric oxide synthase (nNOS), an enzyme implicated in neurodegenerative disorders, is an attractive strategy for treating or preventing these diseases. We previously developed several classes of 2-aminoquinoline-based nNOS inhibitors, but these compounds had drawbacks including off-target promiscuity, low activity against human nNOS, and only modest selectivity for nNOS over related enzymes. In this study, we synthesized new nNOS inhibitors based on 7-phenyl-2-aminoquinoline and assayed them against rat and human nNOS, human eNOS, and murine and (in some cases) human iNOS. Compounds with a meta-relationship between the aminoquinoline and a positively charged tail moiety were potent and had up to nearly 900-fold selectivity for human nNOS over human eNOS. X-ray crystallography indicates that the amino groups of some compounds occupy a water-filled pocket surrounding an nNOS-specific aspartate residue (absent in eNOS). This interaction was confirmed by mutagenesis studies, making 7-phenyl-2-aminoquinolines the first aminoquinolines to interact with this residue.

Activity-Induced SUMOylation of Neuronal Nitric Oxide Synthase Is Associated with Plasticity of Synaptic Transmission and Extracellular Signal-Regulated Kinase 1/2 Signaling.

Aims: Neuronal nitric oxide synthase (nNOS) and nitric oxide (NO) signaling have been implicated in learning, memory, and underlying long-lasting synaptic plasticity. In this study, we aimed at detecting whether nNOS is a target protein of SUMOylation in the hippocampus and its contributions to hippocampal long-term potentiation (LTP) of synaptic transmission. Results: We showed that N-methyl-d-aspartate receptor-dependent neuronal activity enhancement induced the attachment of small ubiquitin-like modifier 1 (SUMO1) to nNOS. Protein inhibitor of activated STAT3 (PIAS3) promoted SUMO1 conjugation at K725 and K739 on nNOS, which upregulated NO production and nNOS S1412 phosphorylation (activation). In addition, the N-terminus (amino acids 43-86) of PIAS3 bound nNOS directly. Tat-tagged PIAS3 segment representing amino acids 43-86, a cell-permeable peptide containing PIAS3 residues 43-86, suppressed activity-induced nNOS SUMOylation by disrupting PIAS3-nNOS association. It also decreased LTP-related expression of Arc and brain-derived neurotrophic factor and blocked signaling via extracellular signal-regulated kinase (ERK) 1/2 and Elk-1 in the hippocampus. More importantly, PIAS3-mediated nNOS SUMOylation was required for activity-regulated ERK1/2 activation in nNOS-positive neurons and hippocampal LTP induction. Innovation and Conclusion: These findings indicated that network activity-regulated nNOS SUMOylation underlies excitatory synaptic LTP by facilitating nNOS-NO-ERK1/2 signal cascades.

Mechanism and regulation of ferrous heme-nitric oxide (NO) oxidation in NO synthases.

Nitric oxide (NO) synthases (NOSs) catalyze the formation of NO from l-arginine. We have shown previously that the NOS enzyme catalytic cycle involves a large number of reactions but can be characterized by a global model with three main rate-limiting steps. These are the rate of heme reduction by the flavin domain (kr ), of dissociation of NO from the ferric heme-NO complex (kd ), and of oxidation of the ferrous heme-NO complex (kox). The reaction of oxygen with the ferrous heme-NO species is part of a futile cycle that does not directly contribute to NO synthesis but allows a population of inactive enzyme molecules to return to the catalytic cycle, and thus, enables a steady-state NO synthesis rate. Previously, we have reported that this reaction does involve the reaction of oxygen with the NO-bound ferrous heme complex, but the mechanistic details of the reaction, that could proceed via either an inner-sphere or an outer-sphere mechanism, remained unclear. Here, we present additional experiments with neuronal NOS (nNOS) and inducible NOS (iNOS) variants (nNOS W409F and iNOS K82A and V346I) and computational methods to study how changes in heme access and electronics affect the reaction. Our results support an inner-sphere mechanism and indicate that the particular heme-thiolate environment of the NOS enzymes can stabilize an N-bound FeIII-N(O)OO- intermediate species and thereby catalyze this reaction, which otherwise is not observed or favorable in proteins like globins that contain a histidine-coordinated heme.

Structural Basis for Isoform Selective Nitric Oxide Synthase Inhibition by Thiophene-2-carboximidamides.

The overproduction of nitric oxide in the brain by neuronal nitric oxide synthase (nNOS) is associated with a number of neurodegenerative diseases. Although inhibiting nNOS is an important therapeutic goal, it is important not to inhibit endothelial NOS (eNOS) because of the critical role played by eNOS in maintaining vascular tone. While it has been possible to develop nNOS selective aminopyridine inhibitors, many of the most potent and selective inhibitors exhibit poor bioavailability properties. Our group and others have turned to more biocompatible thiophene-2-carboximidamide (T2C) inhibitors as potential nNOS selective inhibitors. We have used crystallography and computational methods to better understand how and why two commercially developed T2C inhibitors exhibit selectivity for human nNOS over human eNOS. As with many of the aminopyridine inhibitors, a critical active site Asp residue in nNOS versus Asn in eNOS is largely responsible for controlling selectivity. We also present thermodynamic integration results to better understand the change in p Ka and thus the charge of inhibitors once bound to the active site. In addition, relative free energy calculations underscore the importance of enhanced electrostatic stabilization of inhibitors bound to the nNOS active site compared to eNOS.

A surface magnetic imprinted polymers as artificial receptors for selective and efficient capturing of new neuronal nitric oxide synthase-post synaptic density protein-95 uncouplers.

In this work, surface magnetic molecularly imprinted polymers (SMMIPs) were synthesized and used as artificial receptors in the dispersive magnetic solid phase extraction (DMSPE) for capturing potential neuronal nitric oxide synthase-post synaptic density protein-95 (nNOS-PSD-95) uncouplers, which is known as neuroprotection against stroke. Factors that affected selective separation and adsorption of the artificial receptors, such as the amount of template, the types of functional monomer and porogen solvents, and the molar ratio of template/functional monomer/cross-linker were optimized. The artificial receptors were also characterized using fourier transformed infrared, scanning electron microscope, thermal gravimetric analysis and physical property measurement systems. Multiple interactions between template and SMMIPs led to larger binding capacities, faster binding kinetics, quicker separation abilities and more efficient selectivity than the surface magnetic nonimprinted polymers (SMNIPs). The SMMIPs were successfully applied to capture potential nNOS-PSD-95 uncouplers from complex samples, and eight compounds were seized and confirmed rapidly when combined with HPLC and MS. The detection of the new nNOS-PSD-95 uncouplers ranged from 0.001 to 1.500 mg/mL with correlation coefficients of 0.9990-0.9995. The LOD and LOQ were 0.10-0.68 µg/mL and 0.47-2.11 µg/mL, respectively. The neuroprotective effect and co-immunoprecipitation test in vitro revealed that Emodin-1-O-ß-d-glucoside, Rhaponticin, Gnetol and 2,3,5,4'-Tetrahydroxystilbene-2-O-ß-d-glucoside have neuroprotective and uncoupling activities, and that they may be the new uncouplers of nNOS-PSD-95.

Calmodulin-induced Conformational Control and Allostery Underlying Neuronal Nitric Oxide Synthase Activation.

Nitric oxide synthase (NOS) is the primary generator of nitric oxide signals controlling diverse physiological processes such as neurotransmission and vasodilation. NOS activation is contingent on Ca2+/calmodulin binding at a linker between its oxygenase and reductase domains to induce large conformational changes that orchestrate inter-domain electron transfer. However, the structural dynamics underlying activation of full-length NOS remain ambiguous. Employing hydrogen-deuterium exchange mass spectrometry, we reveal mechanisms underlying neuronal NOS activation by calmodulin and regulation by phosphorylation. We demonstrate that calmodulin binding orders the junction between reductase and oxygenase domains, exposes the FMN subdomain, and elicits a more dynamic oxygenase active site. Furthermore, we demonstrate that phosphorylation partially mimics calmodulin activation to modulate neuronal NOS activity via long-range allostery. Calmodulin binding and phosphorylation ultimately promote a more dynamic holoenzyme while coordinating inter-domain communication and electron transfer.

A cross-domain charge interaction governs the activity of NO synthase.

NO synthase (NOS) enzymes perform interdomain electron transfer reactions during catalysis that may rely on complementary charge interactions at domain-domain interfaces. Guided by our previous results and a computer-generated domain-docking model, we assessed the importance of cross-domain charge interactions in the FMN-to-heme electron transfer in neuronal NOS (nNOS). We reversed the charge of three residues (Glu-762, Glu-816, and Glu-819) that form an electronegative triad on the FMN domain and then individually reversed the charges of three electropositive residues (Lys-423, Lys-620, and Lys-660) on the oxygenase domain (NOSoxy), to potentially restore a cross-domain charge interaction with the triad, but in reversed polarity. Charge reversal of the triad completely eliminated heme reduction and NO synthesis in nNOS. These functions were partly restored by the charge reversal at oxygenase residue Lys-423, but not at Lys-620 or Lys-660. Full recovery of heme reduction was probably muted by an accompanying change in FMN midpoint potential that made electron transfer to the heme thermodynamically unfavorable. Our results provide direct evidence that cross-domain charge pairing is required for the FMN-to-heme electron transfer in nNOS. The unique ability of charge reversal at position 423 to rescue function indicates that it participates in an essential cross-domain charge interaction with the FMN domain triad. This supports our domain-docking model and suggests that it may depict a productive electron transfer complex formed during nNOS catalysis.

cAMP-dependent post-translational modification of neuronal nitric oxide synthase neuroprotects penile erection in rats.

OBJECTIVES: To evaluate neuronal nitric oxide (NO) synthase (nNOS) phosphorylation, nNOS uncoupling, and oxidative stress in the penis and major pelvic ganglia (MPG), before and after the administration of the cAMP-dependent protein kinase A (PKA) agonist colforsin in a rat model of bilateral cavernous nerve injury (BCNI),which mimics nerve injury after prostatectomy. MATERIALS AND METHODS: Adult male Sprague-Dawley rats were divided into BCNI and sham-operated groups. Each group included two subgroups: vehicle and colforsin (0.1 mg/kg/day i.p.). After 3 days, erectile function (intracavernosal pressure) was measured and penis and MPG were collected for molecular analyses of phospho (P)-nNOS (Ser-1412 and Ser-847), total nNOS, nNOS uncoupling, binding of protein inhibitor of nNOS (PIN) to nNOS, gp91phox subunit of NADPH oxidase, active caspase 3, PKA catalytic subunit α (PKA-Cα; by Western blot) and oxidative stress (hydrogen peroxide [H2 O2 ] and superoxide by Western blot and microdialysis method). RESULTS: Erectile function was decreased 3 days after BCNI and normalized by colforsin. nNOS phosphorylation on both positive (Ser-1412) and negative (Ser-847) regulatory sites, and nNOS uncoupling, were increased after BCNI in the penis and MPG, and normalized by colforsin. H2 O2 and total reactive oxygen species production were increased in the penis after BCNI and normalized by colforsin. Protein expression of gp91phox was increased in the MPG after BCNI and was normalized by colforsin treatment. Binding of PIN to nNOS was increased in the penis after BCNI and was normalized by colforsin treatment. Protein expression of active Caspase 3 was increased in the MPG after BCNI and was normalized by colforsin treatment. Protein expression of PKA-Cα was decreased in the penis after BCNI and normalized by colforsin. CONCLUSION: Collectively, BCNI impairs nNOS function in the penis and MPG by mechanisms involving its phosphorylation and uncoupling in association with increased oxidative stress, resulting in erectile dysfunction. PKA activation by colforsin reverses these molecular changes and preserves penile erection in the face of BCNI.

Hydrophilic, Potent, and Selective 7-Substituted 2-Aminoquinolines as Improved Human Neuronal Nitric Oxide Synthase Inhibitors.

Neuronal nitric oxide synthase (nNOS) is a target for development of antineurodegenerative agents. Most nNOS inhibitors mimic l-arginine and have poor bioavailability. 2-Aminoquinolines showed promise as bioavailable nNOS inhibitors but suffered from low human nNOS inhibition, low selectivity versus human eNOS, and significant binding to other CNS targets. We aimed to improve human nNOS potency and selectivity and reduce off-target binding by (a) truncating the original scaffold or (b) introducing a hydrophilic group to interrupt the lipophilic, promiscuous pharmacophore and promote interaction with human nNOS-specific His342. We synthesized both truncated and polar 2-aminoquinoline derivatives and assayed them against recombinant NOS enzymes. Although aniline and pyridine derivatives interact with His342, benzonitriles conferred the best rat and human nNOS inhibition. Both introduction of a hydrophobic substituent next to the cyano group and aminoquinoline methylation considerably improved isoform selectivity. Most importantly, these modifications preserved Caco-2 permeability and reduced off-target CNS binding.

Nitrile in the Hole: Discovery of a Small Auxiliary Pocket in Neuronal Nitric Oxide Synthase Leading to the Development of Potent and Selective 2-Aminoquinoline Inhibitors.

Neuronal nitric oxide synthase (nNOS) inhibition is a promising strategy to treat neurodegenerative disorders, but the development of nNOS inhibitors is often hindered by poor pharmacokinetics. We previously developed a class of membrane-permeable 2-aminoquinoline inhibitors and later rearranged the scaffold to decrease off-target binding. However, the resulting compounds had decreased permeability, low human nNOS activity, and low selectivity versus human eNOS. In this study, 5-substituted phenyl ether-linked aminoquinolines and derivatives were synthesized and assayed against purified NOS isoforms. 5-Cyano compounds are especially potent and selective rat and human nNOS inhibitors. Activity and selectivity are mediated by the binding of the cyano group to a new auxiliary pocket in nNOS. Potency was enhanced by methylation of the quinoline and by introduction of simple chiral moieties, resulting in a combination of hydrophobic and auxiliary pocket effects that yielded high (∼500-fold) n/e selectivity. Importantly, the Caco-2 assay also revealed improved membrane permeability over previous compounds.

Calpain-1 resident in lipid raft/caveolin-1 membrane microdomains plays a protective role in endothelial cells.

We are here reporting that calpain-1 is a constitutive component of a distinct lipid raft/caveolin-1 microdomain isolated from bEnd5 cells in association with endothelial nitric oxide synthase (eNOS) and heat shock protein 90 (HSP90). Perturbations in intracellular calcium concentration by Ca2+-ionophore A23187 or prolonged cell exposure to high glucose induce a significant decrease in the level of eNOS accompanied by a recruitment of additional HSP90 molecules at this site. In these conditions the cells are more resistant to cell death by Ca2+ overload. The decrease of eNOS has been due not only to its Ca2+-mediated release from the caveolin-1 aggregates but also to its digestion by calpain-1. The specific involvement of calpain-1 in digestion of eNOS is supported by the preventive effect of a synthetic calpain inhibitor (CI-2) and by the absence of calpain-2 and calpastatin in the caveolin-1 microdomain. These results suggest that the protein adjustments observed in lipid raft/caveolin-1 microdomains could be visualized as a process required to protect the cells against NO overproduction and aberrant calpain activation. Alterations in eNOS, calpain-1 and HSP90 levels have been observed in aorta of Zucker Diabetic Rats (ZDR). The loss of HSP90 occurring in these animals indicates an aberrant activation of calpain and thereby the transition from a physiological to a pathological cell condition.

The tetrahydrobiopterin radical interacting with high- and low-spin heme in neuronal nitric oxide synthase - A new indicator of the extent of NOS coupling.

Reaction intermediates trapped during the single-turnover reaction of the neuronal ferrous nitric oxide synthase oxygenase domain (Fe(II)nNOSOX) show four EPR spectra of free radicals. Fully-coupled nNOSOX with cofactor (tetrahydrobiopterin, BH4) and substrate (l-arginine) forms the typical BH4 cation radical with an EPR spectrum ~4.0mT wide and hyperfine tensors similar to reports for a biopterin cation radical in inducible NOSOX (iNOSOX). With excess thiol, nNOSox lacking BH4 and l-arg is known to produce superoxide. In contrast, we find that nNOSOX with BH4 but no l-arg forms two radicals with rather different, fast (~250µs at 5K) and slower (~500µs at 20K), electron spin relaxation rates and a combined ~7.0mT wide EPR spectrum. Rapid freeze-quench CW- and pulsed-EPR measurements are used to identify these radicals and their origin. These two species are the same radical with identical nuclear hyperfine couplings, but with spin-spin couplings to high-spin (4.0mT component) or low-spin (7.0mT component) Fe(III) heme. Uncoupled reactions of nNOS leave the enzyme in states that can be chemically reduced to sustain unregulated production of NO and reactive oxygen species in ischemia-reperfusion injury. The broad EPR signal is a convenient indicator of uncoupled nNOS reactions producing low-spin Fe(III) heme.

The expression signatures of neuronal nitric oxide synthase (NOS1) in turbot (Scophthalmus maximus L.) mucosal surfaces against bacterial challenge.

The mucosal surfaces constitute the first immune barrier of host defense and also serve as the dynamic interfaces that simultaneously mediate a diverse array of critical physiological processes. It has been long hypothesized that observed difference of disease resistance among different fish strains and species are strongly correlated to the activities of the immune actors in mucosal surfaces. Particularly, neuronal NOS (nNOS or NOS1) is a constitutively expressed gene that catalyzes the oxidation of l-arginine and water to nitric oxide (NO), which is known as a potent host defence effector in immune system with antimicrobial activity. Moreover, NOS1 was detected to be expressed in fish mucosal surfaces, but its activities in mucosal immune responses were always overlooked. In this regard, we identified the NOS1 of turbot and characterized its expression patterns in mucosal tissues following Vibrio anguillarum and Streptococcus iniae challenge. The results showed that the NOS1 gene had a 4389 bp open reading frame (ORF) that encoded 1462 amino acids. Phylogenetic analysis showed the turbot NOS1 had the strongest relationship to Larimichthys crocea. And the syntenic analysis revealed the similar neighboring genes associated with turbot NOS1, compared with other teleost and mammals. In addition, NOS1 was widely expressed in all examined tissues with the highest expression level in brain, followed by intestine and gill. Finally, the NOS1 showed a general trend of up-regulation in mucosal tissues following both bacterial challenge, with the highest up-regulation in intestine. The significant quick induction of NOS1 in mucosal surfaces against infection indicated its key roles to prevent pathogen attachment and entry in mucosal immunity. More functional studies are needed to conduct in teleost to better understand the roles of NOS1 in maintaining the integrity of the mucosal barriers against infection.

Tetrahydrobiopterin redox cycling in nitric oxide synthase: evidence supports a through-heme electron delivery.

The nitric oxide synthases (NOS) catalyze a two-step oxidation of l-arginine (Arg) to generate NO. In the first step, O2 activation involves one electron being provided to the heme by an enzyme-bound 6R-tetrahydro-l-biopterin cofactor (H4 B), and the H4 B radical must be reduced back to H4 B in order for NOS to continue catalysis. Although an NADPH-derived electron is used to reduce the H4 B radical, how this occurs is unknown. We hypothesized that the NOS flavoprotein domain might reduce the H4 B radical by utilizing the NOS heme porphyrin as a conduit to deliver the electron. This model predicts that factors influencing NOS heme reduction should also influence the extent and rate of H4 B radical reduction in kind. To test this, we utilized single catalytic turnover and stop-freeze methods, along with electron paramagnetic resonance spectroscopy, to measure the rate and extent of reduction of the 5-methyl-H4 B radical formed in neuronal NOS (nNOS) during Arg hydroxylation. We used several nNOS variants that supported either a slower or faster than normal rate of ferric heme reduction. We found that the rates and extents of nNOS heme reduction correlated well with the rates and extents of 5-methyl-H4 B radical reduction among the various nNOS enzymes. This supports a model where the heme porphyrin transfers an electron from the NOS flavoprotein to the H4 B radical formed during catalysis, revealing that the heme plays a dual role in catalyzing O2 activation or electron transfer at distinct points in the reaction cycle.

Exploring second coordination sphere effects in nitric oxide synthase.

Second coordination sphere (SCS) effects in proteins are modulated by active site residues and include hydrogen bonding, electrostatic/dipole interactions, steric interactions, and π-stacking of aromatic residues. In Cyt P450s, extended H-bonding networks are located around the proximal cysteinate ligand of the heme, referred to as the 'Cys pocket'. These hydrogen bonding networks are generally believed to regulate the Fe-S interaction. Previous work identified the S(Cys) → Fe σ CT transition in the high-spin (hs) ferric form of Cyt P450cam and corresponding Cys pocket mutants by low-temperature (LT) MCD spectroscopy [Biochemistry 50:1053, 2011]. In this work, we have investigated the effect of the hydrogen bond from W409 to the axial Cys ligand of the heme in the hs ferric state (with H4B and L-Arg bound) of rat neuronal nitric oxide synthase oxygenase construct (nNOSoxy) using MCD spectroscopy. For this purpose, wt enzyme and W409 mutants were investigated where the H-bonding network with the axial Cys ligand is perturbed. Overall, the results are similar to Cyt P450cam and show the intense S(Cys) → Fe σ CT band in the LT MCD spectrum at about 27,800 cm-1, indicating that this feature is a hallmark of {heme-thiolate} active sites. The discovery of this MCD feature could constitute a new approach to classify {heme-thiolate} sites in hs ferric proteins. Finally, the W409 mutants show that the hydrogen bond from this group only has a small effect on the Fe-S(Cys) bond strength, at least in the hs ferric form of the protein studied here. Low-temperature MCD spectroscopy is used to investigate the effect of the hydrogen bond from W409 to the axial Cys ligand of the heme in neuronal nitric oxide synthase. The intense S(Cys) → Fe σ-CT band is monitored to identify changes in the Fe-S(Cys) bond in wild-type protein and W409 mutants.