Nitric oxide synthase

Nitric oxide synthases (

NOS catalyzes the reaction:[3]

• 2 L-arginine + 3 NADPH + 3 H⁺ + 4 O₂ ${\displaystyle \rightleftharpoons }$ 2 citrulline +2 nitric oxide + 4 H₂O + 3 NADP⁺

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:

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⁺ 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.^[6]

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.^[7]

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 episodes (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.^[8]

Classification

Different members of the NOS family are encoded by separate genes.^[9] There are three known isoforms in mammals, two are constitutive (cNOS) and the third is inducible (iNOS).^[10] Cloning of NOS enzymes indicates that cNOS include both brain constitutive (NOS1) and endothelial constitutive (NOS3); the third is the inducible (NOS2) gene.^[10] Recently, NOS activity has been demonstrated in several bacterial species, including the notorious pathogens Bacillus anthracis and Staphylococcus aureus.^[11]

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

nNOS

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.^[14]

The gene coding for nNOS is located on Chromosome 12.^[15]

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 Ca²⁺. The gene coding for iNOS is located on Chromosome 17.

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.^[17]

It has been suggested that pathologic generation of

Endothelial NOS (eNOS), also known as nitric oxide synthase 3 (NOS3), generates NO in blood vessels and is involved with regulating vascular function. The gene coding for eNOS is located on Chromosome 7.^[15] A constitutive Ca²⁺ 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.^[19] eNOS localization to endothelial membranes is mediated by cotranslational N-terminal myristoylation and post-translational palmitoylation.^[20]

bNOS

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.^[21][22]

Chemical reaction

Nitric oxide synthases produce NO by catalysing a five-electron oxidation of a guanidino nitrogen of L-arginine (L-Arg). Oxidation of L-Arg to L-citrulline occurs via two successive monooxygenation reactions producing N^ω-hydroxy-L-arginine (NOHLA) as an intermediate. 2 mol of O₂ and 1.5 mol of NADPH are consumed per mole of NO formed.^[3]

Structure

The enzymes exist as homodimers. In eukaryotes, each monomer consisting of two major regions: an N-terminal

In NOS1 and NOS3, physiological concentrations of Ca²⁺ in cells regulate the binding of calmodulin to the "latch domains", thereby initiating electron transfer from the flavins to the heme moieties. In contrast, calmodulin remains tightly bound to the inducible and Ca²⁺-insensitive isoform (iNOS or NOS2) even at a low intracellular Ca²⁺ 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.^[24] 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]}. 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]}. The rate-limiting step for the production of nitric oxide may well be the availability of L-arginine in some cell types. This may be particularly important after the induction of NOS2.

Inhibitors

• Biological functions of nitric oxide
• Nitric-oxide synthase (NAD(P)H-dependent)
• Nitric oxide synthase 2 (inducible)

References