Editing Nitric oxide synthase

{{Short description|Class of enzymes}}
{{cleanup|reason=Has an ontology problem. Do we want to describe all NO synthases, or just the one with EC code 1.14.13.39?|date=March 2025}}
{{infobox enzyme
| Name       = Nitric-oxide synthase (NADPH)
| EC_number  = 1.14.13.39
| CAS_number = 125978-95-2
| GO_code    = 0051767
| image      = 1nsi.png
| width      =
| caption    = Human inducible nitric oxide synthase. PDB {{PDBe|1nsi}} 
}}
{{Infobox protein family
| Symbol = NO_synthase 
| Name = Nitric oxide synthase, oxygenase domain 
| image = Nitric_Oxide_Synthase.png
| width = 
| caption = Structure of endothelial nitric oxide synthase heme domain.<ref name="pmid21138269">{{PDB|3N5P}}; {{cite journal |vauthors=Delker SL, Xue F, Li H, Jamal J, Silverman RB, Poulos TL | title = Role of zinc in isoform-selective inhibitor binding to neuronal nitric oxide synthase | journal = Biochemistry | volume = 49 | issue = 51 | pages = 10803–10 |date=December 2010 | pmid = 21138269 | doi = 10.1021/bi1013479 | pmc=3193998}}</ref>
| Pfam= PF02898
| InterPro= IPR004030
| SMART= 
| Prosite =          
| SCOP = 1nos
| TCDB = 
| OPM family= 
| OPM protein= 
}} 
'''Nitric oxide synthases''' ('''NOSs''') are a family of [[enzymes]] catalyzing the production of [[nitric oxide]] (NO) from [[L-arginine]]. NO is an important [[biological functions of nitric oxide|cellular signaling]] molecule. It helps modulate [[vascular tone]], [[insulin]] secretion, airway tone, and [[peristalsis]], and is involved in [[angiogenesis]] and neural development. It may function as a retrograde [[neurotransmitter]]. Nitric oxide is mediated in mammals by the [[calcium in biology|calcium]]-[[calmodulin]] controlled [[isozyme|isoenzyme]]s eNOS ([[endothelial NOS]]) and nNOS (neuronal NOS).<ref>{{Cite journal |last1=Ahmad |first1=Nashrah |last2=Ansari |first2=Mohammad Y. |last3=Haqqi |first3=Tariq M. |date=October 2020 |title=Role of iNOS in osteoarthritis: Pathological and therapeutic aspects |journal=Journal of Cellular Physiology |language=en |volume=235 |issue=10 |pages=6366–6376 |doi=10.1002/jcp.29607 |issn=0021-9541 |pmc=8404685 |pmid=32017079}}</ref> The inducible isoform, iNOS, involved in immune response, binds [[calmodulin]] at physiologically relevant concentrations, and produces NO as an immune defense mechanism, as NO is a free radical with an unpaired electron. It is the [[proximate and ultimate causation|proximate cause]] of [[septic shock]] and may function in [[autoimmunity|autoimmune]] disease.

In the context of [[eukaryote]] biology, ''nitric oxide synthase'' refers to '''nitric-oxide synthase (NADPH)''' ({{EC number|1.14.13.39}}), which catalyzes the reaction:<ref name="pmid7510950" />
* 2 L-[[arginine]] + 3 [[nicotinamide adenine dinucleotide phosphate|NADPH]] +  3 H<sup>+</sup> + 4 O<sub>2</sub> <math>\rightleftharpoons</math> 2 [[citrulline]] +2  [[nitric oxide]] + 4 H<sub>2</sub>O + 3 NADP<sup>+</sup>

NOS isoforms catalyze other leak and side reactions, such as [[superoxide]] production at the expense of NADPH. As such, this stoichiometry is not generally observed, and reflects the three electrons supplied per NO by NADPH.

Eukaryotic NOS isozymes are catalytically self-sufficient. The electron flow is: [[NADPH]] → [[flavin adenine dinucleotide|FAD]] → [[Flavin mononucleotide|FMN]] → [[heme]] → [[dioxygen|O<sub>2</sub>]]. [[Tetrahydrobiopterin]] provides an additional electron during the catalytic cycle which is replaced during turnover. [[Zinc]], though not a cofactor, also participates but as a structural element.<ref name="pmid25180171">{{cite journal|vauthors=Cortese-Krott M, Kulakov L, Opländer C, Kolb-Bachofen V, Kröncke K, Suschek C |title=Zinc regulates iNOS-derived nitric oxide formation in endothelial cells |journal=Redox Bio. J. |volume=2014 |issue=2 |pages=945–954 |date=July 2014 |doi=10.1016/j.redox.2014.06.011 |pmid=25180171 |pmc=4143817}}</ref> NOSs are unique in that they use five [[Cofactor (biochemistry)|cofactor]]s and are the only known [[enzyme]] that binds [[flavin adenine dinucleotide]] (FAD), [[flavin mononucleotide]] (FMN), [[heme]], [[tetrahydrobiopterin]] (BH<sub>4</sub>) and [[calmodulin]].{{citation needed|date=May 2016}}

The EC number 1.14.13.39 specifically refers to synthases with linked oxygenase and reductase domains, i.e. "catalytically self-sufficient" NO synthases. This kind of synthase is ound in eukaryotes and, through independent domain acquisition, ''[[Sorangium cellulosum]]''. Most bacteria and archaea have a version that only has an oxidase domain and depend on a partner protein; these are categorized as EC 1.14.14.47 "[[nitric-oxide synthase (flavodoxin)]]". All these enzymes' oxygenase domains share a common ancestor (see "oxygenase domain" infobox).<ref>{{cite journal |last1=Agapie |first1=Theodor |last2=Suseno |first2=Sandy |last3=Woodward |first3=Joshua J. |last4=Stoll |first4=Stefan |last5=Britt |first5=R. David |last6=Marletta |first6=Michael A. |title=NO formation by a catalytically self-sufficient bacterial nitric oxide synthase from Sorangium cellulosum |journal=Proceedings of the National Academy of Sciences |date=22 September 2009 |volume=106 |issue=38 |pages=16221–16226 |doi=10.1073/pnas.0908443106|doi-access=free |pmid=19805284 |pmc=2752531 |bibcode=2009PNAS..10616221A }}</ref><ref>{{cite web |title=ExplorEnz: EC 1.14.13.39 |url=https://www.enzyme-database.org/query.php?ec=1.14.13.39 |website=www.enzyme-database.org}}</ref>

== Species distribution ==

Arginine-derived NO synthesis has been identified in mammals, fish, birds, invertebrates, and bacteria.<ref name="pmid8782597">{{cite book |vauthors=Liu Q, Gross SS | title = Nitric Oxide Part A: Sources and Detection of NO; NO Synthase | chapter = Binding sites of nitric oxide synthases | series = Methods in Enzymology | volume = 268 | pages = 311–24 | year = 1996 | pmid = 8782597 | doi = 10.1016/S0076-6879(96)68033-1 | isbn = 9780121821692 }}</ref> 

Best studied are mammals, where three distinct genes encode NOS [[isozyme]]s: [[neuronal]] (nNOS or NOS-1), [[cytokine]]-inducible (iNOS or NOS-2) and [[endothelial]] (eNOS or NOS-3). iNOS and nNOS are soluble and found predominantly in the [[cytosol]], while eNOS is membrane associated. Evidence has been found for NO signaling in plants, but plant genomes are devoid of homologs to the superfamily which generates NO in other kingdoms.<ref name="pmid7510950">{{cite journal |vauthors=Knowles RG, Moncada S | title = Nitric oxide synthases in mammals | journal = Biochem. J. | volume = 298 | issue = 2| pages = 249–58 |date=March 1994 | doi = 10.1042/bj2980249 | pmid = 7510950 | pmc = 1137932 }}</ref>

== Chemical reaction ==
[[Image:NOSreaction.svg]]

Nitric oxide synthases produce NO by catalysing a five-electron oxidation of a guanidino nitrogen of <small>L</small>-arginine (<small>L</small>-Arg). Oxidation of <small>L</small>-Arg to <small>L</small>-citrulline occurs via two successive monooxygenation reactions producing ''N''<sup>ω</sup>-hydroxy-<small>L</small>-arginine (NOHLA) as an intermediate. 2&nbsp;mol of O<sub>2</sub> and 1.5&nbsp;mol of NADPH are consumed per mole of NO formed.<ref name="pmid7510950"/>

== Function ==

In mammals, the endothelial isoform is the primary signal generator in the control of vascular tone, insulin secretion, and [[airway tone]], is involved in regulation of cardiac function and angiogenesis (growth of new blood vessels). NO produced by eNOS has been shown to be a vasodilator identical to the [[endothelium-derived relaxing factor]] produced in response to shear from increased blood flow in arteries.  This dilates blood vessels by relaxing smooth muscle in their linings. eNOS is the primary controller of smooth muscle tone. NO activates [[guanylate cyclase]], which induces smooth muscle relaxation by:
* Increased intracellular cGMP, which inhibits [[calcium]] entry into the cell, and decreases intracellular calcium concentrations
* Activation of K<sup>+</sup> channels, which leads to hyperpolarization and relaxation
* Stimulates a cGMP-dependent protein [[kinase]] that activates [[myosin]] light chain phosphatase, the enzyme that dephosphorylates [[myosin]] light chains, which leads to smooth muscle relaxation.

eNOS plays a critical role in embryonic heart development and morphogenesis of coronary arteries and cardiac valves.<ref name="pmid22579300">{{cite journal |vauthors=Liu Y, Feng Q | title = NOing the heart: role of nitric oxide synthase-3 in heart development | journal = Differentiation | volume = 84 | issue = 1 | pages = 54–61 |date=July 2012 | pmid = 22579300 | doi = 10.1016/j.diff.2012.04.004 }}</ref>
 
The neuronal isoform is involved in the development of nervous system. It functions as a retrograde neurotransmitter important in long term potentiation and hence is likely to be important in memory and learning. nNOS has many other physiological functions, including regulation of cardiac function and peristalsis and sexual arousal in males and females.  An alternatively spliced form of nNOS is a major muscle protein that produces signals in response to calcium release from the SR. nNOS in the heart protects against cardiac arrhythmia induced by myocardial infarction.<ref name="pmid19770398">{{cite journal |vauthors=Burger DE, Lu X, Lei M, Xiang FL, Hammoud L, Jiang M, Wang H, Jones DL, Sims SM, Feng Q | title = Neuronal nitric oxide synthase protects against myocardial infarction-induced ventricular arrhythmia and mortality in mice | journal = Circulation | volume = 120 | issue = 14 | pages = 1345–54 |date=October 2009 | pmid = 19770398 | doi = 10.1161/CIRCULATIONAHA.108.846402 | doi-access = free }}</ref>
 
The primary receiver for NO produced by eNOS and nNOS is soluble guanylate cyclase, but many secondary targets have been identified. S-nitrosylation appears to be an important mode of action.

The inducible isoform iNOS produces large amounts of NO as a defense mechanism. It is synthesized by many cell types in response to cytokines and is an important factor in the response of the body to attack by parasites, bacterial infection, and tumor growth.  It is also  the cause of [[septic shock]] and may play a role in many diseases with an autoimmune etiology.

NOS signaling is involved in development and in fertilization in vertebrates.  It has been implicated in transitions between vegetative and reproductive states in invertebrates, and in differentiation leading to spore formation in slime molds. NO produced by bacterial NOS is protective against oxidative damage.

NOS activity has also been correlated with [[major depressive episode]]s (MDEs) in the context of [[major depressive disorder]], in a large case-control treatment study published in mid-2021. 460 patients with a current major depressive episode were compared to 895 healthy patients, and by measuring L-citrulline/L-arginine ratio before and after 3–6 months of antidepressant treatment, results indicate that patients in a major depressive episode have significantly lower NOS activity compared to healthy patients, whilst treatment with antidepressants significantly elevated NOS activity levels in patients in a major depressive episode.<ref>{{cite journal|url=https://www.cambridge.org/core/journals/psychological-medicine/article/abs/nitric-oxide-synthase-activity-in-major-depressive-episodes-before-and-after-antidepressant-treatment-results-of-a-large-casecontrol-treatment-study/55DB96AEB6B05D0E9E792A4D97FD0A6C#|author1=E. Loeb|author2=K. El Asmar|journal=[[Psychological Medicine]]|author3=S. Trabado|author4=F. Gressier|author5=R. Colle|author6=A. Rigal|author7=S. Martin|author8=C. Verstuyft|author9=B. Fève|author10=P. Chanson|author11=L. Becquemont|author12=E. Corruble|title=Nitric Oxide Synthase activity in major depressive episodes before and after antidepressant treatment: Results of a large case-control treatment study|date=January 2022|accessdate=26 December 2021|volume=52|issue=1|pages=80–89|doi=10.1017/S0033291720001749|pmid=32524920|s2cid=219587961|url-access=subscription}}</ref>

== Mammalian isoforms ==

Different members of the NOS family are encoded by separate genes.<ref name="pmid9366709">{{cite journal |vauthors=Taylor BS, Kim YM, Wang Q, Shapiro RA, Billiar TR, Geller DA | title = Nitric oxide down-regulates hepatocyte-inducible nitric oxide synthase gene expression | journal = Arch Surg | volume = 132 | issue = 11 | pages = 1177–83 |date=November 1997 | pmid = 9366709 | doi = 10.1001/archsurg.1997.01430350027005}}</ref> There are three known isoforms in mammals, two are constitutive (cNOS) and the third is inducible (iNOS).<ref name="pmid10320659">{{cite journal | author = Stuehr DJ | title = Mammalian nitric oxide synthases | journal = Biochim. Biophys. Acta | volume = 1411 | issue = 2–3 | pages = 217–30 |date=May 1999 | pmid = 10320659 | doi = 10.1016/S0005-2728(99)00016-X| doi-access =  }}</ref> Cloning of NOS enzymes indicates that cNOS include both brain constitutive ([[NOS1]]) and endothelial constitutive ([[endothelial NOS|NOS3]]); the third is the inducible ([[Nitric oxide synthase 2 (inducible)|NOS2]]) gene.<ref name="pmid10320659"/> Recently, NOS activity has been demonstrated in several bacterial species, including the notorious pathogens Bacillus anthracis and Staphylococcus aureus.<ref name="pmid18316370">{{cite journal |vauthors=Gusarov I, Starodubtseva M, Wang ZQ, McQuade L, Lippard SJ, Stuehr DJ, Nudler E | title = Bacterial Nitric-oxide Synthases Operate without a Dedicated Redox Partner | journal = J. Biol. Chem. | volume = 283 | issue = 19 | pages = 13140–7 |date=May 2008 | pmid = 18316370 | pmc = 2442334 | doi = 10.1074/jbc.M710178200 | doi-access = free }}</ref>

The different forms of NO synthase have been classified as follows:

{| class="wikitable"
! Name || Gene(s) || Location || width=30% | Function
 |-
 | '''[[Neuron]]al NOS''' (nNOS or NOS1)  || [[NOS1]] (Chromosome 12) ||
*[[nervous tissue]]
*[[skeletal muscle]] type II
||
* multiple functions (see below)
 |-
 | '''Inducible NOS''' (iNOS or NOS2) 
Calcium insensitive 
|| [[Nitric oxide synthase 2 (inducible)|NOS2]] (Chromosome 17) ||
*[[immune system]]
*[[cardiovascular system]]
||
*[[immune]] defense against pathogens
 |-
 | '''[[Endothelial NOS]]''' (eNOS or NOS3 or cNOS)  || [[Endothelial NOS|NOS3]] (Chromosome 7) ||
*[[endothelium]]
||
*[[vasodilation]]
|}

=== nNOS ===

[[Neuron]]al NOS (nNOS) produces NO in [[nervous tissue]] in both the central and peripheral [[nervous systems]]. Its functions include:<ref>{{cite journal|last1=Förstermann|first1=Ulrich|last2=Sessa|first2=William|title=Nitric oxide synthases: regulation and function|journal=European Heart Journal|date=Apr 2012|volume=33|issue=7|pages=829–837|pmc=3345541|doi=10.1093/eurheartj/ehr304|pmid=21890489}}</ref>

* Synaptic plasticity in the central nervous system (CNS)
* Smooth muscle relaxation
* Central regulation of blood pressure
* Vasodilatation via peripheral nitrergic nerves

Neuronal NOS also performs a role in cell communication and is associated with plasma membranes. nNOS action can be inhibited by NPA ([[N-propyl-L-arginine]]). This form of the enzyme is specifically inhibited by [[7-Nitroindazole|7-nitroindazole]].<ref name="pmid8619882">{{cite journal |vauthors=Southan GJ, Szabó C | title = Selective pharmacological inhibition of distinct nitric oxide synthase isoforms | journal = Biochem. Pharmacol. | volume = 51 | issue = 4 | pages = 383–94 |date=February 1996 | pmid = 8619882 | doi = 10.1016/0006-2952(95)02099-3 }}</ref>

The subcellular localisation of nNOS in skeletal muscle is mediated by anchoring of nNOS to [[dystrophin]]. nNOS contains an additional N-terminal domain, the [[PDZ domain]].<ref name="pmid7535955">{{cite journal |vauthors=Ponting CP, Phillips C | title = DHR domains in syntrophins, neuronal NO synthases and other intracellular proteins | journal = Trends Biochem. Sci. | volume = 20 | issue = 3 | pages = 102–3 |date=March 1995 | pmid = 7535955 | doi = 10.1016/S0968-0004(00)88973-2 }}</ref>

The gene coding for nNOS is located on Chromosome 12.<ref name="Nitric oxide synthases in mammals">{{cite journal |vauthors=Knowles RG, Moncada S | title = Nitric oxide synthases in mammals | journal = Biochem. J. | volume = 298 | issue = 2| pages = 249–58 |date=March 1994 | pmid = 7510950 | pmc = 1137932 | doi =  10.1042/bj2980249}}</ref>

=== iNOS ===

As opposed to the critical calcium-dependent regulation of constitutive NOS enzymes (nNOS and eNOS), iNOS has been described as calcium-insensitive, likely due to its tight non-covalent interaction with calmodulin (CaM) and Ca<sup>2+</sup>. The gene coding for iNOS is located on Chromosome 17.<ref name="Nitric oxide synthases in mammals" /> While evidence for ‘baseline’ iNOS expression has been elusive, [[IRF1]] and [[NF-κB]]-dependent activation of the inducible NOS promoter supports an inflammation mediated stimulation of this transcript. iNOS produces large quantities of  NO upon stimulation, such as by [[proinflammatory cytokine]]s (e.g. [[Interleukin 1 family|Interleukin-1]], [[Tumor necrosis factor alpha]] and [[Interferon gamma]]).<ref name="pmid7537721">{{cite journal |vauthors=Green SJ, Scheller LF, Marletta MA, Seguin MC, Klotz FW, Slayter M, Nelson BJ, Nacy CA | title = Nitric oxide: cytokine-regulation of nitric oxide in host resistance to intracellular pathogens | journal = Immunol. Lett. | volume = 43 | issue = 1–2 | pages = 87–94 |date=December 1994 | pmid = 7537721 | doi = 10.1016/0165-2478(94)00158-8| hdl = 2027.42/31140 | url = https://deepblue.lib.umich.edu/bitstream/2027.42/31140/1/0000037.pdf | hdl-access = free }}</ref>

Induction of the high-output iNOS usually occurs in an oxidative environment, and thus high levels of NO have the opportunity to react with [[superoxide]] leading to [[peroxynitrite]] formation and cell toxicity. These properties may define the roles of iNOS in host immunity, enabling its participation in anti-microbial and anti-tumor activities as part of the oxidative burst of macrophages.<ref name="pmid12379825">{{cite journal |vauthors=Mungrue IN, Husain M, Stewart DJ | title = The role of NOS in heart failure: lessons from murine genetic models | journal = Heart Fail Rev | volume = 7 | issue = 4 | pages = 407–22 |date=October 2002 | pmid = 12379825 | doi = 10.1023/a:1020762401408| s2cid = 26600958 }}</ref>

It has been suggested that pathologic generation of [[nitric oxide]] through increased iNOS production may decrease [[fallopian tube|tubal]] [[cilia]]ry beats and smooth muscle contractions and thus affect embryo transport, which may consequently result in [[ectopic pregnancy]].<ref name="pmid19482272">{{cite journal |vauthors=Al-Azemi M, Refaat B, Amer S, Ola B, Chapman N, Ledger W | title = The expression of inducible nitric oxide synthase in the human fallopian tube during the menstrual cycle and in ectopic pregnancy | journal = Fertil. Steril. | volume = 94 | issue = 3 | pages = 833–40 |date=August 2010 | pmid = 19482272 | doi = 10.1016/j.fertnstert.2009.04.020 }}</ref>

=== eNOS ===
{{Main|Endothelial NOS}}
Endothelial NOS (eNOS), also known as nitric oxide synthase 3 (NOS3), generates NO in [[blood vessel]]s and is involved with regulating vascular function. The gene coding for eNOS is located on Chromosome 7.<ref name="Nitric oxide synthases in mammals" /> A constitutive Ca<sup>2+</sup> dependent NOS provides a basal release of NO. eNOS localizes to caveolae, a plasma membrane domain primarily composed of the protein [[caveolin 1]], and to the Golgi apparatus. These two eNOS populations are distinct, but are both necessary for proper NO production and cell health.<ref name ="pmid32152543">{{cite journal |vauthors = Maulik SJ, Junyi Z, Aneesh TV, Yamuna K | title = A DNA-based fluorescent probe maps NOS3 activity with subcellular spatial resolution
| journal = Nat. Chem. Biol. | issue = 6 | pages = 660–6 | date=March 2020 | volume = 16
| pmid = 32152543 | doi = 10.1038/s41589-020-0491-3| s2cid = 212642840
}}</ref> eNOS localization to endothelial membranes is mediated by cotranslational N-terminal [[myristoylation]] and post-translational [[palmitoylation]].<ref name="pmid9199168">{{cite journal |vauthors=Liu J, Hughes TE, Sessa WC | title = The First 35 Amino Acids and Fatty Acylation Sites Determine the Molecular Targeting of Endothelial Nitric Oxide Synthase into the Golgi Region of Cells: A Green Fluorescent Protein Study | journal = J. Cell Biol. | volume = 137 | issue = 7 | pages = 1525–35 |date=June 1997 | pmid = 9199168 | pmc = 2137822 | doi = 10.1083/jcb.137.7.1525 }}</ref> As an essential co-factor for nitric oxide synthase, [[tetrahydrobiopterin]] (BH4) supplementation has shown beneficial results for the treatment of [[endothelial dysfunction]] in animal experiments and clinical trials, although the tendency of BH4 to become oxidized to BH2 remains a problem.<ref name="pmid29596860">{{cite journal | vauthors = Yuyun MF, Ng LL, Ng GA | title=Endothelial dysfunction, endothelial nitric oxide bioavailability, tetrahydrobiopterin, and 5-methyltetrahydrofolate in cardiovascular disease. Where are we with therapy? | journal= Microvascular Research | volume=119 | pages=7–12 | year=2018 | doi= 10.1016/j.mvr.2018.03.012 | pmid=29596860}}</ref>

== Structure ==

The enzymes exist as homodimers. In eukaryotes, each monomer consisting of two major regions: an N-terminal [[oxygenase]] domain, which belongs to the class of heme-thiolate proteins, and a multi-domain C-terminal [[reductase]], which is homologous to NADPH:[[cytochrome P450 reductase]] ({{EC number|1.6.2.4}}) and other flavoproteins. The FMN binding domain is homologous to flavodoxins, and the two domain fragment containing the FAD and NADPH binding sites is homologous to flavodoxin-NADPH reductases. The interdomain linker between the oxygenase and reductase domains contains a [[calmodulin]]-binding sequence. The oxygenase domain is a unique extended beta sheet cage with binding sites for heme and pterin.

NOSs can be [[protein dimer|dimeric]], calmodulin-dependent or calmodulin-containing [[cytochrome p450]]-like [[hemoprotein]] that combines reductase and oxygenase catalytic domains in one dimer, bear both [[flavin adenine dinucleotide]] (FAD) and [[flavin mononucleotide]] (FMN), and carry out a 5`-electron oxidation of non-aromatic [[amino acid]] arginine with the aid of tetrahydrobiopterin.<ref name="pmid9493011">{{cite journal |vauthors=Chinje EC, Stratford IJ | title = Role of nitric oxide in growth of solid tumours: a balancing act | journal = Essays Biochem. | volume = 32 | pages = 61–72 | year = 1997 | pmid = 9493011 }}</ref>

All three [[isoform]]s (each of which is presumed to function as a [[homodimer]] during activation) share a carboxyl-terminal reductase domain homologous to the [[cytochrome P450 reductase]]. They also share an amino-terminal [[oxygenase domain]] containing a [[heme]] [[prosthetic group]], which is linked in the middle of the [[protein]] to a [[calmodulin]]-binding domain. Binding of calmodulin appears to act as a "molecular switch" to enable [[electron]] flow from flavin prosthetic groups in the reductase domain to heme. This facilitates the conversion of O<sub>2</sub> and <small>L</small>-arginine to [[nitric oxide|NO]] and <small>L</small>-citrulline. The oxygenase domain of each NOS isoform also contains an BH<sub>4</sub> prosthetic group, which is required for the efficient generation of NO. Unlike other enzymes where BH<sub>4</sub> is used as a source of reducing equivalents and is recycled by [[dihydrobiopterin reductase]] ({{EC number|1.5.1.33}}), BH<sub>4</sub> activates heme-bound O<sub>2</sub> by donating a single electron, which is then recaptured to enable nitric oxide release.

The first nitric oxide synthase to be identified was found in neuronal tissue (NOS1 or nNOS); the [[endothelial]] NOS (eNOS or NOS3) was the third to be identified. They were originally classified as "constitutively expressed" and "Ca<sup>2+</sup> sensitive" but it is now known that they are present in many different [[cell (biology)|cell]] types and that expression is regulated under specific physiological conditions.

In NOS1 and NOS3, physiological concentrations of Ca<sup>2+</sup> in cells regulate the binding of calmodulin to the "latch domains", thereby initiating electron transfer from the [[Flavin group|flavins]] to the [[heme]] moieties. In contrast, calmodulin remains tightly bound to the inducible and Ca<sup>2+</sup>-insensitive isoform (iNOS or NOS2) even at a low intracellular Ca<sup>2+</sup> activity, acting essentially as a subunit of this isoform.

Nitric oxide may itself regulate NOS expression and activity. Specifically, NO has been shown to play an important [[negative feedback]] regulatory role on NOS3, and therefore vascular endothelial cell function.<ref>{{Cite journal|last1=Kopincová|first1=Jana|last2=Púzserová|first2=Angelika|last3=Bernátová|first3=Iveta|date=2011-06-01|title=Biochemical aspects of nitric oxide synthase feedback regulation by nitric oxide|journal=Interdisciplinary Toxicology|language=en|volume=4|issue=2|pages=63–8|doi=10.2478/v10102-011-0012-z|issn=1337-9569|pmc=3131676|pmid=21753901}}</ref> This process, known formally as ''S''-nitrosation (and referred to by many in the field as ''S''-nitrosylation), has been shown to reversibly inhibit NOS3 activity in vascular endothelial cells. This process may be important because it is regulated by cellular redox conditions and may thereby provide a mechanism for the association between "oxidative stress" and endothelial dysfunction. In addition to NOS3, both NOS1 and NOS2 have been found to be ''S''-nitrosated, but the evidence for dynamic regulation of those NOS isoforms by this process is less complete{{Citation needed|date=December 2014}}. In addition, both NOS1 and NOS2 have been shown to form ferrous-nitrosyl complexes in their heme prosthetic groups that may act partially to self-inactivate these enzymes under certain conditions{{Citation needed|date=December 2014}}. The rate-limiting step for the production of nitric oxide may well be the availability of <small>L</small>-arginine in some cell types. This may be particularly important after the [[Enzyme induction and inhibition|induction]] of NOS2.

==Inhibitors==
[[Ronopterin]] (VAS-203), also known as 4-amino-tetrahydrobiopterin (4-ABH<sub>4</sub>), an [[structural analog|analogue]] of BH<sub>4</sub> (a cofactor of NOS), is an NOS inhibitor that is under development as a [[neuroprotective agent]] for the treatment of [[traumatic brain injury]].[http://adisinsight.springer.com/drugs/800023224] Other NOS inhibitors that have been or are being researched for possible clinical use include [[cindunistat]], [[A-84643]], [[ONO-1714]], [[L-NOARG]], [[NCX-456]], [[VAS-2381]], [[GW-273629]], [[NXN-462]], [[CKD-712]], [[KD-7040]], and [[guanidinoethyldisulfide]], [https://pubchem.ncbi.nlm.nih.gov/compound/2733516 TFPI] among others.

== In non-mammal eukaryotes ==
NOS is found in many groups of eukaryotes (metazoans, fungi, algae, plants), mostly sharing the fused oxygenase-CAM-flavin-FNR structure of mammalian NOS.<ref name="pmid30468649"/> There can, however, be important changes to the structure. For example, a number of algal NOS has a C-terminal [[globin]] domain, which could confer additional functionality.<ref>{{cite journal |last1=Chatelain |first1=P |last2=Astier |first2=J |last3=Wendehenne |first3=D |last4=Rosnoblet |first4=C |last5=Jeandroz |first5=S |title=Identification of Partner Proteins of the Algae Klebsormidium nitens NO Synthases: Toward a Better Understanding of NO Signaling in Eukaryotic Photosynthetic Organisms. |journal=Frontiers in Plant Science |date=2021 |volume=12 |pages=797451 |doi=10.3389/fpls.2021.797451 |doi-access=free |pmid=35003186|pmc=8728061 }}</ref>

== In prokaryotes ==

{| class="wikitable"
! Name || Gene(s) || Location || width=30% | Function
|-
 | '''Bacterial NOS''' (bNOS)  || multiple ||
*[[Gram-positive bacteria]]<br/>(various)
||
*defense against [[oxidative stress]], [[antibiotic]]s, [[immune system|immune attack]]
|}
Bacterial NOS (bNOS) has been shown to protect bacteria against oxidative stress, diverse antibiotics, and host immune response. bNOS plays a key role in the transcription of [[superoxide dismutase]] (SodA). Bacteria late in the log phase who do not possess bNOS fail to upregulate SodA, which disables the defenses against harmful oxidative stress. Initially, bNOS may have been present to prepare the cell for stressful conditions but now seems to help shield the bacteria against conventional antimicrobials. As a clinical application, a bNOS inhibitor could be produced to decrease the load of Gram positive bacteria.<ref name="pmid16172391">{{cite journal |vauthors=Gusarov I, Nudler E | title = NO-mediated cytoprotection: Instant adaptation to oxidative stress in bacteria | journal = Proc. Natl. Acad. Sci. U.S.A. | volume = 102 | issue = 39 | pages = 13855–60 |date=September 2005 | pmid = 16172391 | pmc = 1236549 | doi = 10.1073/pnas.0504307102 | bibcode = 2005PNAS..10213855G | doi-access = free }}</ref><ref name="pmid19745150">{{cite journal |vauthors=Gusarov I, Shatalin K, Starodubtseva M, Nudler E | title = Endogenous Nitric Oxide Protects Bacteria Against a Wide Spectrum of Antibiotics | journal = Science | volume = 325 | issue = 5946 | pages = 1380–4 |date=September 2009 | pmid = 19745150 | pmc = 2929644 | doi = 10.1126/science.1175439  | bibcode = 2009Sci...325.1380G }}</ref>

They seem to have a similar function in archaea.<ref>{{cite journal |last1=Orsini |first1=SS |last2=James |first2=KL |last3=Reyes |first3=DJ |last4=Couto-Rodriguez |first4=RL |last5=Gulko |first5=MK |last6=Witte |first6=A |last7=Carroll |first7=RK |last8=Rice |first8=KC |title=Bacterial-like nitric oxide synthase in the haloalkaliphilic archaeon Natronomonas pharaonis. |journal=MicrobiologyOpen |date=November 2020 |volume=9 |issue=11 |pages=e1124 |doi=10.1002/mbo3.1124 |pmid=33306280|pmc=7658456 }}</ref>

They exist in a wide variety of bacteria, including Gram negative ones (the aforementioned ''Sorangium cellulosum'' is Gram negative). The fused [Fe-S] NOS is by no means rare among bacteria, being seen across Cyanobacteria and Proteobacteria.<ref name="pmid30468649">{{cite journal |last1=Santolini |first1=J |title=What does "NO-Synthase" stand for ? |journal=Frontiers in Bioscience (Landmark Edition) |date=1 January 2019 |volume=24 |issue=1 |pages=133–171 |doi=10.2741/4711 |pmid=30468649}}</ref>

==See also ==
* [[Biological functions of nitric oxide]]

==References==
{{notelist}}
{{Reflist|2}}

==External links==
* {{MeshName|Nitric+oxide+synthase}}
* [http://nobelprize.org/nobel_prizes/medicine/laureates/1998/ The Nobel Prize in Physiology or Medicine 1998]
* University of Edinburgh, School of Chemistry - [https://web.archive.org/web/20130212143027/http://homepages.ed.ac.uk/sd01/nospage.htm NO Synthase]
* [http://www.proteopedia.org/wiki/index.php/Nitric_oxide_synthase/ Nitric Oxide Synthase in Proteopedia]

{{Neurotransmitter metabolism enzymes}}
{{Dioxygenases}}
{{Enzymes}}
{{Nitric oxide signaling}}
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{{DEFAULTSORT:Nitric Oxide Synthase}}
[[Category:EC 1.14.13]]
[[Category:Heme-thiolate proteins]]