Nitric Oxide: Synthesis and Action


Nitric oxide (NO) is a diatomic radical implicated in a variety of pathways including vascular homeostasis, neurotransmission and immune defence. NO is produced endogenously by three nitric oxide synthases (NOS) and can also be formed from nitrates and nitrites. Several mechanisms regulate NOS function, including protein–protein interactions, posttranslational modifications, cell localization, calcium levels, substrate availability, and the cell redox status. Picomolar NO concentrations activate guanylyl cyclase to increase cGMP levels and lead to vasodilation and memory formation. Excess NO concentrations cause posttranslational modifications of cellular components and formation of peroxynitrite leading to inflammation and cell death. Due to its involvement in multiple pathways and high reactivity with a myriad of downstream targets, tight regulation of NO production is crucial to maintain cellular function. Major advancements in understanding NO chemistry, production and downstream signalling have provided new avenues to target this pathway for the treatment of cardiovascular, neurodegenerative and autoimmune diseases.

Key Concepts

  • Nitric oxide is produced endogenously by three nitric oxide synthase (NOS) isozymes.
  • Low NO levels mediate cell signalling, while high NO levels mediate the immune response and defence against pathogens.
  • A tight regulation of NO production is necessary to maintain cellular function.
  • The guanylyl cyclase enzyme (GC1) is the primary receptor of NO in smooth muscle cells and mediates vasorelaxation. Better understanding of NO/GC1 signalling will lead to novel drugs targeting this pathway for improved cardiovascular health.
  • Nitrite, a product of nitrate reduction via oral bacteria, is a biologically relevant source of NO under hypoxic and acidic conditions.
  • Dysfunction in NO synthesis due to oxidative damage leads to NOS uncoupling and increased output of ROS, a well‐known trait of cardiovascular disease (CVD).

Keywords: nitric oxide; nitric oxide synthase; guanylyl cyclase; nitrate; nitrite; cardiovascular diseases

Figure 1. Overall mechanism and architecture of NOS enzymes. (a) General mechanism for NO production. (b) The overall architecture of NOS enzymes is very similar and contains an N‐terminal NOS oxygenase module (NOSox), an intervening Ca2+/calmodulin binding region (CaM) and a C‐terminal NOS reductase module (NOSred). (c) Specific features of each NOS isoform include a PDZ domain for NOSI, myristoylation and palmitoylation sites in NOSII. All three isoforms contain a C‐terminal tail (CT). NOSI and NOSIII also contain an autoinhibitory helix (AH) and a CD2A inhibitory element, as well as various phosphorylation sites. (d) X‐ray structure of the human NOSII oxygenase module. (e) X‐ray structure of Ca2+/CaM bound to a NOSIII‐derived peptide. (f) X‐ray structure of the rat NOSI reductase module.
Figure 2. Overall NOS pathways and regulation. (a) NOSI becomes activated with increased Ca2+ concentrations, phosphorylation and protein–protein interactions. NOSI‐derived NO improves blood flow and increases neuronal cell growth (1). Hyperactivated NOSI can lead to excitotoxicity and apoptosis (2). NOSI activity is shutdown through phosphorylation and proteasomal degradation (3). (b) NOSII expression is induced by various cytokines. Immune cells such as macrophages and neutrophils use NOSII to generate cytotoxic levels of NO to kill invading pathogens (1). Excessive NOSII activity can damage healthy cells leading to inflammation and cell death (2). NOSII is neutralised through proteasomal degradation and a negative feedback loop with NF‐κB (3). (c) NOSIII is activated similarly to NOSI. A variety of kinases, protein–protein interactions and increased Ca2+ levels enhance NOSIII activity. NOSIII‐derived NO inhibits platelet aggregation and dilates neighbouring blood vessels (1). Reactive oxygen species (ROS) and low BH4 levels can uncouple NOSIII activity and lead to superoxide formation, which in turn decreases available NO and exacerbates oxidative stress (2). Phosphorylation and proteasomal degradation turn this pathway off (3).
Figure 3. The NO–GC1–cGMP pathway and therapeutic targets. NO is generated in the endothelium and diffuses to smooth muscle cells. GC1 (blue) is the primary target for NO binding, which triggers an increase in cGMP generation. Second messenger cGMP activates PKG, which phosphorylates downstream targets for improved blood flow. Reactive oxygen species (ROS) and phosphodiesterase‐5 (PDE5) can inhibit this pathway (red arrows). Several pharmacological targets for this pathway are shown with green arrows.
Figure 4. The nitrate/nitrite/NO pathway. Nitrate is obtained primarily in the diet from leafy greens and cured meats. Oral bacteria oxidise nitrate to nitrite, which acts as an important source of NO under hypoxic and acidic conditions.


Alderton WK, Cooper CE and Knowles RG (2001) Nitric oxide synthases: structure, function and inhibition. Biochemical Journal 357: 593–615.

Aoyagi M, Arvai AS, Tainer JA and Getzoff ED (2003) Structural basis for endothelial nitric oxide synthase binding to calmodulin. EMBO Journal 22: 766–775.

Arnold WP, Mittal CK, Katsuki S and Murad F (1977) Nitric oxide activates guanylate cyclase and increases guanosine 3':5'‐cyclic monophosphate levels in various tissue preparations. Proceedings of the National Academy of Sciences of the United States of America 74: 3203–3207.

Bauer PM, Fulton D, Boo YC, et al. (2003) Compensatory phosphorylation and protein‐protein interactions revealed by loss of function and gain of function mutants of multiple serine phosphorylation sites in endothelial nitric‐oxide synthase. Journal of Biological Chemistry 278: 14841–14849.

Bogdan C (2015) Nitric oxide synthase in innate and adaptive immunity: an update. Trends in Immunology 36: 161–178.

Boo YC, Hwang J, Sykes M, et al. (2002) Shear stress stimulates phosphorylation of eNOS at Ser 635 by a protein kinase A‐dependent mechanism. American Journal of Physiology – Heart and Circulatory Physiology 283: H1819–H1828.

Butt E, Bernhardt M, Smonleski A, et al. (2000) Endothelial nitric‐oxide synthase (type III) is activated and becomes calcium independent upon phosphorylation by cyclic nucleotide‐dependent protein kinases. Journal of Biological Chemistry 275: 5179–5187.

Campbell MG, Smith BC, Potter CS, Carragher B and Marletta MA (2014) Molecular architecture of mammalian nitric oxide synthases. Proceedings of the National Academy of Sciences 111: E3614–E3623.

Crane BR, Arvai AS, Gachhui R, et al. (1997) The structure of nitric oxide synthase oxygenase domain and inhibitor complexes. Science 278: 425–431.

Crane BR, Arvai AS, Ghosh DK, et al. (1998) Structure of nitric oxide synthase oxygenase dimer with pterin and substrate. Science 279: 2121–2126.

Crane BR, Sudhamsu J and Patel BA (2010) Bacterial nitric oxide synthases. Annual Review of Biochemistry 79: 445–470.

Dimmeler S, Fleming I, Fisslthaler B, et al. (1999) Activation of nitric oxide synthase in endothelial cells by Akt‐dependent phosphorylation. Nature 399: 601–605.

Feron O, Michel JB, Sase K and Michel T (1998) Dynamic regulation of endothelial nitric oxide synthase: complementary roles of dual acylation and caveolin interactions †. Biochemistry 37: 193–200.

Fischmann TO, Hruza A, Niu XD, et al. (1999) Structural characterization of nitric oxide synthase isoforms reveals striking active‐site conservation. Nature Structural Biology 6: 233–242.

Fisslthaler B, Loot AE, Mohamed A, Busse R and Fleming I (2008) Inhibition of endothelial nitric oxide synthase activity by proline‐rich tyrosine kinase 2 in response to fluid shear stress and insulin. Circulation Research 102: 1520–1528.

Flinspach M, Li H, Jamal J, et al. (2004) Structural basis for dipeptide amide isoform‐selective inhibition of neuronal nitric oxide synthase. Nature Structural & Molecular Biology 11: 54–59.

Forstermann U and Sessa WC (2012) Nitric oxide synthases: regulation and function. European Heart Journal 33: 829–837.

Fulton D, Gratton J‐P, Mccabe T‐J, et al. (1999) Regulation of endothelium‐derived nitric oxide production by the protein kinase Akt. Nature 399: 597–601.

Fulton D, Ruan L, Sood SG, et al. (2008) Agonist‐stimulated endothelial nitric oxide synthase activation and vascular relaxation: role of eNOS phosphorylation at Tyr83. Circulation Research 102: 497–504.

Fulton DJR (2016) Transcriptional and posttranslational regulation of eNOS in the endothelium. In: Advances in Pharmacology, pp. 29–64. Elsevier.

Furchgott RF (1996) The discovery of endothelium‐derived relaxing factor and its importance in the identification of nitric oxide. JAMA 276: 1186–1188.

Gallis B, Corthals GL, Goodlett DR, et al. (1999) Identification of flow‐dependent endothelial nitric‐oxide synthase phosphorylation sites by mass spectrometry and regulation of phosphorylation and nitric oxide production by the phosphatidylinositol 3‐kinase inhibitor LY294002. Journal of Biological Chemistry 274: 30101–30108.

Garcin ED, Bruns CM, Lloyd SJ, et al. (2004) Structural basis for isozyme‐specific regulation of electron transfer in nitric oxide synthase. Journal of Biological Chemistry 279: 37918–37927.

Ghofrani H‐A, Humbert M, Langleben D, et al. (2017) Riociguat: mode of action and clinical development in pulmonary hypertension. Chest 151: 468–480.

Guo FH, Comhair SAA, Zheng S, et al. (2000) Molecular mechanisms of increased nitric oxide (NO) in asthma: evidence for transcriptional and post‐translational regulation of NO synthesis. Journal of Immunology 164: 5970.

Heiss E and Dirsch V (2014) Regulation of eNOS enzyme activity by posttranslational modification. Current Pharmaceutical Design 20: 3503–3513.

Hickok J and Thomas D (2010) Nitric oxide and cancer therapy: the emperor has NO clothes. Current Pharmaceutical Design 16: 381–391.

Ignarro LJ, Buga GM, Wood KS, Byrns RE and Chaudhuri G (1987) Endothelium‐derived relaxing factor produced and released from artery and vein is nitric oxide. Proceedings of the National Academy of Sciences of the United States of America 84: 9265–9269.

Lundberg JO, Gladwin MT and Weitzberg E (2015) Strategies to increase nitric oxide signalling in cardiovascular disease. Nature Reviews Drug Discovery 14: 623–641.

Michell BJ, Harris MB, Chen ZP, et al. (2002) Identification of regulatory sites of phosphorylation of the bovine endothelial nitric‐oxide synthase at serine 617 and serine 635. Journal of Biological Chemistry 277: 42344–42351.

Montfort WR, Wales JA and Weichsel A (2017) Structure and activation of soluble guanylyl cyclase, the nitric oxide sensor. Antioxidants & Redox Signaling 26: 107–121.

Palmer RMJ, Ferrige AG and Moncada S (1987) Nitric oxide release accounts for the biological activity of endothelium‐derived relaxing factor. Nature 327: 524–526.

Piazza M, Futrega K, Spratt DE, Dieckmann T and Guillemette JG (2012) Structure and dynamics of calmodulin (CaM) bound to nitric oxide synthase peptides: effects of a phosphomimetic CaM mutation. Biochemistry 51: 3651–3661.

Poulos TL and Li H (2017) Nitric oxide synthase and structure‐based inhibitor design. Nitric Oxide 63: 68–77.

Raman CS, Li H, Martasek P, et al. (1998) Crystal structure of constitutive endothelial nitric oxide synthase: a paradigm for pterin function involving a novel metal center. Cell 95: 939–950.

Rameau GA, Chiu L and Ziff EB (2004) Bidirectional regulation of neuronal nitric‐oxide synthase phosphorylation at serine 847 by the N‐methyl‐D‐aspartate receptor. Journal of Biological Chemistry 279: 14307–14314.

Rameau GA, Tukey DS, Garcin‐Hosfield ED, et al. (2007) Biphasic coupling of neuronal nitric oxide synthase phosphorylation to the NMDA receptor regulates AMPA receptor trafficking and neuronal cell death. Journal of Neuroscience 27: 3445–3455.

Salerno JC, Ray K, Poulos T, Li H and Ghosh DK (2013) Calmodulin activates neuronal nitric oxide synthase by enabling transitions between conformational states. FEBS Letters 587: 44–47.

Santolini J (2011) The molecular mechanism of mammalian NO‐synthases: a story of electrons and protons. Journal of Inorganic Biochemistry 105: 127–141.

Shiva S (2013) Nitrite: a physiological store of nitric oxide and modulator of mitochondrial function. Redox Biology 1: 40–44.

Socco S, Bovee RC, Palczewski MB, Hickok JR and Thomas DD (2017) Epigenetics: the third pillar of nitric oxide signaling. Pharmacological Research 121: 52–58.

Song T, Hatano N, Horii M, et al. (2004) Calcium/calmodulin‐dependent protein kinase I inhibits neuronal nitric‐oxide synthase activity through serine 741 phosphorylation. FEBS Letters 570: 133–137.

Stuehr DJ (1997) Structure‐function aspects in the nitric oxide synthases. Annual Review of Pharmacology and Toxicology 37: 339–359.

Venema RC, Sayegh HS, Kent JD and Harrison DG (1996) Identification, characterization, and comparison of the calmodulin‐binding domains of the endothelial and inducible nitric oxide synthases. Journal of Biological Chemistry 271: 6435–6440.

Volkmann N, Martásek P, Roman LJ, et al. (2014) Holoenzyme structures of endothelial nitric oxide synthase – an allosteric role for calmodulin in pivoting the FMN domain for electron transfer. Journal of Structural Biology 188: 46–54.

Weitzberg E and Lundberg JO (2013) Novel aspects of dietary nitrate and human health. Annual Review of Nutrition 33: 129–159.

Xia C, Misra I, Iyanagi T and Kim J‐JP (2009) Regulation of Interdomain Interactions by calmodulin in inducible nitric‐oxide synthase. Journal of Biological Chemistry 284: 30708–30717.

Yokom AL, Morishima Y, Lau M, et al. (2014) Architecture of the nitric‐oxide synthase holoenzyme reveals large conformational changes and a calmodulin‐driven release of the FMN domain. Journal of Biological Chemistry 289: 16855–16865.

Zhang J, Martasek P, Paschke R, et al. (2001) Crystal structure of the FAD/NADPH‐binding domain of rat neuronal nitric‐oxide synthase. Comparisons with NADPH‐cytochrome P450 oxidoreductase. Journal of Biological Chemistry 276: 37506–37513.

Zhang YH (2016) Neuronal nitric oxide synthase in hypertension – an update. Journal of Clinical Hypertension 22: 20.

Further Reading

Brian SB, Bian K and Murad M (2009) Discovery of the nitric oxide signaling pathway and targets for drug development. Front. Biosci. (Landmark Ed) 14: 1–18.

Fukuto JM, Carrington SJ, Harrison JG, et al. (2012) Small molecules signaling agents: the integrated chemistry and biochemistry of nitrogen oxides, oxides of carbon, dioxygen, hydrogen sulfide, and their derived species. Chem Res Toxicol. 25 (4): 769–793.

Ghimire K, Altmann HM, Straub AC and Isenberg JS (2017) NItric Oxide. What's new to NO? Am J. Physiol Cell Physiol. 312 (3): C254–C262.

Haque MM, Ray SS and Stuehr DJ (2016) Phosphorylation controls endothelial nitric‐oxide synthase by regulating its conformational dynamics. J. Biol. Chem. 291 (4): 23047–23057.

Kraehling JR and Sessa WC (2017) Contemporary approaches to modulating the nitric oxide‐cGMP pathway in cardiovascular disease. Circ Res. 120 (7): 1174–1182.

National Academy of Sciences (2000) Beyond discovery: from explosives to the gas that heals.

Sarti P (2013) Nitric oxide in human health and disease. In: eLS. Chichester: John Wiley & Sons, Ltd. ISBN: 10.1002/9780470015902.a0003390.pub2.

Stuehr DJ and Vasquez‐Vivar J (2017) Nitric oxide synthases‐from genes to function. Nitric oxide 63: 29.

Wink DA, Molon B, Agnelllini AHR, et al. (2002) Immune defence: role of reactive nitrogen intermediates. In: eLS. Chichester: John Wiley & Sons, Ltd. ISBN: 10.1038/npg.els.0000484.

Zhao Y, Vanhoutte PM and Leung SW (2015) Vascular nitric oxide: beyond eNOS. J. Pharmacol. Sci. 129 (2): 83–94.

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Childers, Kenneth C, and Garcin, Elsa D(Sep 2017) Nitric Oxide: Synthesis and Action. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0000634.pub2]