Nitric oxide synthase
Encyclopedia
Nitric oxide synthases (NOSs) are a family of enzymes that catalyze the production of nitric oxide
(NO) from L-arginine. NO is an important cellular signaling
molecule, having a vital role in many biological processes. It is the intercellular signal that controls vascular tone (hence blood pressure), insulin
secretion, airway tone, and peristalsis
, and is involved in angiogenesis
(growth of new blood vessels) and in the development of nervous system. It is believed to function as a retrograde neurotransmitter
and hence is likely to be important in learning. Nitric oxide signalling is mediated in mammals by the calcium
/calmodulin
controlled isoenzyme
s eNOS (endothelial NOS) and nNOS (neuronal NOS); the inducible isoform iNOS is involved in immune response, binds calmodulin at all physiologically relevant concentrations, and produces large amounts of NO as a defense mechanism. It is the proximate cause of septic shock
and may play a role in many diseases with an autoimmune
etiology.
The canonical reaction catalyzed by NOS is:
NOS isoforms catalyze many other leak and side reactions such as superoxide
production at the expense of NADPH, so this stoichiometry is not generally observed. The unusual stoichiometry reflects the three electrons supplied per NO by NADPH; NO is a free radical with an unpaired electron.
NOSs are unusual in that they require five cofactor
s. Eukaryotic NOS isozymes are catalytically self-sufficient. The electron flow in the NO synthase reaction is: NADPH --> FAD
--> FMN
--> heme
--> O2. Tetrahydrobiopterin provides an additional electron during the catalytic cycle which is replaced during turnover. ). NOS is the only known enzyme
that binds flavin adenine dinucleotide (FAD), flavin mononucleotide
(FMN), heme
, tetrahydrobiopterin
(BH4) and calmodulin
.
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.
(NO
) ), 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:
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 from of nNOS is a major muscle protein that produces signals in response to calcium release from the SR.
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 synthisized 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.
) and endothelial constitutive (NOS3
), the third is the inducible (NOS2) gene. Recently, NOS activity has been demonstrated in several bacterial species, including such notorious pathogens as Bacillus anthracis and Staphylococcus aureus.
The different forms of NO synthase have been classified as follows:
al NOS (nNOS) produces NO in nervous tissue
in both the central and peripheral nervous system
. 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
.
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
.
and NF-κB-dependent activation of the inducible NOS promoter supports an inflammation mediated stimulation of this transcript.
From a functional perspective, it is important to recognize that 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.
It has been suggested that pathologic generation of nitric oxide
through increased iNOS production may decrease tubal ciliary beats and smooth muscle contractions and thus affect embryo transport, which may consequently result in ectopic pregnancy
.
s and is involved with regulating vascular function. A constitutive Ca2+ dependent NOS provides a basal release of NO. eNOS is associated with plasma membranes surrounding cells and the membranes of Golgi bodies within cells. eNOS localisation to endothelial membranes is mediated by cotranslational N-terminal myristoylation
and post-translational palmitoylation
.
(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.
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 Nω-hydroxy-L-arginine (NOHLA) as an intermediate. 2 mol of O2 and 1.5 mol of NADPH are consumed per mole of NO formed.
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
and other flavoproteins. The FMN binding domain ins 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 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.
All three isoforms (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 O2 and L-arginine to NO
and L-citrulline. The oxygenase domain of each NOS isoform also contains an BH4 prosthetic group, which is required for the efficient generation of NO. Unlike other enzymes where BH4 is used as a source of reducing equivalents and is recycled by dihydrobiopterin reductase
, BH4 activates heme-bound O2 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 "Ca2+ sensitive" but it is now known that they are present in many different cell
types and that expression is regulated under specific physiological conditions.
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. 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. 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. 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.
Nitric oxide
Nitric oxide, also known as nitrogen monoxide, is a diatomic molecule with chemical formula NO. It is a free radical and is an important intermediate in the chemical industry...
(NO) from L-arginine. NO is an important cellular signaling
Biological functions of nitric oxide
The gas nitric oxide plays a wide variety of roles in biological organisms.-Nitric oxide synthesis:Nitric oxide synthases synthesize the metastable free radical nitric oxide . There are three isoforms of the NOS enzyme: endothelial , neuronal , and inducible - each with separate functions...
molecule, having a vital role in many biological processes. It is the intercellular signal that controls vascular tone (hence blood pressure), insulin
Insulin
Insulin is a hormone central to regulating carbohydrate and fat metabolism in the body. Insulin causes cells in the liver, muscle, and fat tissue to take up glucose from the blood, storing it as glycogen in the liver and muscle....
secretion, airway tone, and peristalsis
Peristalsis
Peristalsis is a radially symmetrical contraction and relaxation of muscles which propagates in a wave down the muscular tube, in an anterograde fashion. In humans, peristalsis is found in the contraction of smooth muscles to propel contents through the digestive tract. Earthworms use a similar...
, and is involved in angiogenesis
Angiogenesis
Angiogenesis is the physiological process involving the growth of new blood vessels from pre-existing vessels. Though there has been some debate over terminology, vasculogenesis is the term used for spontaneous blood-vessel formation, and intussusception is the term for the formation of new blood...
(growth of new blood vessels) and in the development of nervous system. It is believed to function as a retrograde neurotransmitter
Neurotransmitter
Neurotransmitters are endogenous chemicals that transmit signals from a neuron to a target cell across a synapse. Neurotransmitters are packaged into synaptic vesicles clustered beneath the membrane on the presynaptic side of a synapse, and are released into the synaptic cleft, where they bind to...
and hence is likely to be important in learning. Nitric oxide signalling is mediated in mammals by the calcium
Calcium in biology
Calcium plays a pivotal role in the physiology and biochemistry of organisms and the cell. It plays an important role in signal transduction pathways, where it acts as a second messenger, in neurotransmitter release from neurons, contraction of all muscle cell types, and fertilization...
/calmodulin
Calmodulin
Calmodulin is a calcium-binding protein expressed in all eukaryotic cells...
controlled isoenzyme
Isozyme
Isozymes are enzymes that differ in amino acid sequence but catalyze the same chemical reaction. These enzymes usually display different kinetic parameters Isozymes (also known as isoenzymes) are enzymes that differ in amino acid sequence but catalyze the same chemical reaction. These enzymes...
s eNOS (endothelial NOS) and nNOS (neuronal NOS); the inducible isoform iNOS is involved in immune response, binds calmodulin at all physiologically relevant concentrations, and produces large amounts of NO as a defense mechanism. It is the proximate cause of septic shock
Septic shock
Septic shock is a medical emergency caused by decreased tissue perfusion and oxygen delivery as a result of severe infection and sepsis, though the microbe may be systemic or localized to a particular site. It can cause multiple organ dysfunction syndrome and death...
and may play a role in many diseases with an autoimmune
Autoimmunity
Autoimmunity is the failure of an organism to recognize its own constituent parts as self, which allows an immune response against its own cells and tissues. Any disease that results from such an aberrant immune response is termed an autoimmune disease...
etiology.
The canonical reaction catalyzed by NOS is:
- L-arginineArginineArginine is an α-amino acid. The L-form is one of the 20 most common natural amino acids. At the level of molecular genetics, in the structure of the messenger ribonucleic acid mRNA, CGU, CGC, CGA, CGG, AGA, and AGG, are the triplets of nucleotide bases or codons that codify for arginine during...
+ 3/2 NADPHNicotinamide adenine dinucleotide phosphateNicotinamide adenine dinucleotide phosphate, abbreviated NADP or TPN in older notation , is a coenzyme used in anabolic reactions, such as lipid and nucleic acid synthesis, which require NADPH as a reducing agent....
+ H+ + 2 O2 = citrullineCitrullineThe organic compound citrulline is an α-amino acid. Its name is derived from citrullus, the Latin word for watermelon, from which it was first isolated in 1930.It has the idealized formula H2NCNH3CHCO2H...
+ nitric oxideNitric oxideNitric oxide, also known as nitrogen monoxide, is a diatomic molecule with chemical formula NO. It is a free radical and is an important intermediate in the chemical industry...
+ 3/2 NADP+
NOS isoforms catalyze many other leak and side reactions such as superoxide
Superoxide
A superoxide, also known by the obsolete name hyperoxide, is a compound that possesses the superoxide anion with the chemical formula O2−. The systematic name of the anion is dioxide. It is important as the product of the one-electron reduction of dioxygen O2, which occurs widely in nature...
production at the expense of NADPH, so this stoichiometry is not generally observed. The unusual stoichiometry reflects the three electrons supplied per NO by NADPH; NO is a free radical with an unpaired electron.
NOSs are unusual in that they require five cofactor
Cofactor (biochemistry)
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the protein's biological activity. These proteins are commonly enzymes, and cofactors can be considered "helper molecules" that assist in biochemical transformations....
s. Eukaryotic NOS isozymes are catalytically self-sufficient. The electron flow in the NO synthase reaction is: NADPH --> FAD
FAD
In biochemistry, flavin adenine dinucleotide is a redox cofactor involved in several important reactions in metabolism. FAD can exist in two different redox states, which it converts between by accepting or donating electrons. The molecule consists of a riboflavin moiety bound to the phosphate...
--> FMN
FMN
FMN may refer to several things:* Flavin mononucleotide.* Forsvarsministeriet, the Danish Ministry of Defence.* Four Corners Regional Airport outside Farmington, New Mexico, its IATA airport code.* Fluid Music....
--> heme
Heme
A heme or haem is a prosthetic group that consists of an iron atom contained in the center of a large heterocyclic organic ring called a porphyrin. Not all porphyrins contain iron, but a substantial fraction of porphyrin-containing metalloproteins have heme as their prosthetic group; these are...
--> O2. Tetrahydrobiopterin provides an additional electron during the catalytic cycle which is replaced during turnover. ). NOS is the only known enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
that binds flavin adenine dinucleotide (FAD), flavin mononucleotide
Flavin mononucleotide
Flavin mononucleotide , or riboflavin-5′-phosphate, is a biomolecule produced from riboflavin by the enzyme riboflavin kinase and functions as prosthetic group of various oxidoreductases including NADH dehydrogenase as well as cofactor in biological blue-light photo receptors...
(FMN), heme
Heme
A heme or haem is a prosthetic group that consists of an iron atom contained in the center of a large heterocyclic organic ring called a porphyrin. Not all porphyrins contain iron, but a substantial fraction of porphyrin-containing metalloproteins have heme as their prosthetic group; these are...
, tetrahydrobiopterin
Tetrahydrobiopterin
Tetrahydrobiopterin or sapropterin is a naturally occurring essential cofactor of the three aromatic amino acid hydroxylase enzymes, used in the degradation of amino acid phenylalanine and in the biosynthesis of the neurotransmitters serotonin , melatonin, dopamine, norepinephrine ,...
(BH4) and calmodulin
Calmodulin
Calmodulin is a calcium-binding protein expressed in all eukaryotic cells...
.
Species distribution
Arginine-derived NO synthesis has been identified in mammals, fish, birds, invertebrates, and bacteria. Best studied are mammals, where three distinct genes encode NOS isozymeIsozyme
Isozymes are enzymes that differ in amino acid sequence but catalyze the same chemical reaction. These enzymes usually display different kinetic parameters Isozymes (also known as isoenzymes) are enzymes that differ in amino acid sequence but catalyze the same chemical reaction. These enzymes...
s: neuronal (nNOS or NOS-1), cytokine
Cytokine
Cytokines are small cell-signaling protein molecules that are secreted by the glial cells of the nervous system and by numerous cells of the immune system and are a category of signaling molecules used extensively in intercellular communication...
-inducible (iNOS or NOS-2) and endothelial (eNOS or NOS-3). iNOS and nNOS are soluble and found predominantly in the cytosol
Cytosol
The cytosol or intracellular fluid is the liquid found inside cells, that is separated into compartments by membranes. For example, the mitochondrial matrix separates the mitochondrion into compartments....
, 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.
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 ( see nitric oxideEndothelium-derived relaxing factor
Endothelium-derived relaxing factor is produced and released by the endothelium to promote smooth muscle relaxation. The best-characterized is nitric oxide . Some sources equate EDRF and nitric oxide....
(NO
Nitric oxide
Nitric oxide, also known as nitrogen monoxide, is a diatomic molecule with chemical formula NO. It is a free radical and is an important intermediate in the chemical industry...
) ), 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
Guanylate cyclase
-Reaction:Guanylate cyclase catalyzes the reaction of guanosine triphosphate to 3',5'-cyclic guanosine monophosphate and pyrophosphate:-Types:...
, which induces smooth muscle relaxation by:
- Increased intracellular cGMP, which inhibits calciumCalciumCalcium is the chemical element with the symbol Ca and atomic number 20. It has an atomic mass of 40.078 amu. Calcium is a soft gray alkaline earth metal, and is the fifth-most-abundant element by mass in the Earth's crust...
entry into the cell, and decreases intracellular calcium concentrations - Activation of K+ channels, which leads to hyperpolarization and relaxation
- Stimulates a cGMP-dependent protein kinaseKinaseIn chemistry and biochemistry, a kinase is a type of enzyme that transfers phosphate groups from high-energy donor molecules, such as ATP, to specific substrates, a process referred to as phosphorylation. Kinases are part of the larger family of phosphotransferases...
that activates myosinMyosinMyosins comprise a family of ATP-dependent motor proteins and are best known for their role in muscle contraction and their involvement in a wide range of other eukaryotic motility processes. They are responsible for actin-based motility. The term was originally used to describe a group of similar...
light chain phosphatase, the enzyme that dephosphorylates myosinMyosinMyosins comprise a family of ATP-dependent motor proteins and are best known for their role in muscle contraction and their involvement in a wide range of other eukaryotic motility processes. They are responsible for actin-based motility. The term was originally used to describe a group of similar...
light chains, which leads to smooth muscle relaxation.
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 from of nNOS is a major muscle protein that produces signals in response to calcium release from the SR.
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 synthisized 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.
Classification
Different members of the NOS family are encoded by separate genes. NOS is one of the most regulated enzymes in biology. There are three known isoforms, two are constitutive (cNOS) and the third is inducible (iNOS). Cloning of NOS enzymes indicates that, cNOS include both brain constitutive (NOS1NOS1
Nitric oxide synthase 1 , also known as NOS1, is an enzyme that in humans is encoded by the NOS1 gene.- Function :Nitric oxide is a messenger molecule with diverse functions throughout the body...
) and endothelial constitutive (NOS3
Endothelial NOS
Endothelial NOS , also known as nitric oxide synthase 3 or constitutive NOS , is an enzyme that in humans is encoded by the NOS3 gene....
), the third is the inducible (NOS2) gene. Recently, NOS activity has been demonstrated in several bacterial species, including such notorious pathogens as Bacillus anthracis and Staphylococcus aureus.
The different forms of NO synthase have been classified as follows:
| Name | Gene(s) | Location | Function >- | Neuron Neuron A neuron is an electrically excitable cell that processes and transmits information by electrical and chemical signaling. Chemical signaling occurs via synapses, specialized connections with other cells. Neurons connect to each other to form networks. Neurons are the core components of the nervous... al NOS (nNOS or NOS1) |
NOS1 NOS1 Nitric oxide synthase 1 , also known as NOS1, is an enzyme that in humans is encoded by the NOS1 gene.- Function :Nitric oxide is a messenger molecule with diverse functions throughout the body... |
|
NOS2 |
|
NOS3 Endothelial NOS Endothelial NOS , also known as nitric oxide synthase 3 or constitutive NOS , is an enzyme that in humans is encoded by the NOS3 gene.... |
|
Vasodilation Vasodilation refers to the widening of blood vessels resulting from relaxation of smooth muscle cells within the vessel walls, particularly in the large arteries, smaller arterioles and large veins. The process is essentially the opposite of vasoconstriction, or the narrowing of blood vessels. When... |
multiple Multiple The word multiple can refer to:*Multiple , multiples of numbers*List of multiple discoveries, instances of scientists, working independently of each other, reaching similar findings... |
|
Oxidative stress Oxidative stress represents an imbalance between the production and manifestation of reactive oxygen species and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage... , antibiotic Antibiotic An antibacterial is a compound or substance that kills or slows down the growth of bacteria.The term is often used synonymously with the term antibiotic; today, however, with increased knowledge of the causative agents of various infectious diseases, antibiotic has come to denote a broader range of... s, immune attack Immune system An immune system is a system of biological structures and processes within an organism that protects against disease by identifying and killing pathogens and tumor cells. It detects a wide variety of agents, from viruses to parasitic worms, and needs to distinguish them from the organism's own... |
nNOS
NeuronNeuron
A neuron is an electrically excitable cell that processes and transmits information by electrical and chemical signaling. Chemical signaling occurs via synapses, specialized connections with other cells. Neurons connect to each other to form networks. Neurons are the core components of the nervous...
al NOS (nNOS) produces NO in nervous tissue
Nervous tissue
Nervous tissue is one of four major classes of vertebrate tissue.Nervous tissue is the main component of the nervous system - the brain, spinal cord, and nerves-which regulates and controls body functions...
in both the central and peripheral nervous system
Nervous system
The nervous system is an organ system containing a network of specialized cells called neurons that coordinate the actions of an animal and transmit signals between different parts of its body. In most animals the nervous system consists of two parts, central and peripheral. The central nervous...
. 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
N-propyl-L-arginine
N-Propyl-L-arginine, or more properly NG-propyl-L-arginine , is a selective inhibitor of neuronal nitric oxide synthase ....
). This form of the enzyme is specifically inhibited by 7-nitroindazole
7-Nitroindazole
7-Nitroindazole, or 7-NI, is a heterocyclic small molecule containing an indazole ring that has been nitrated at the 7 position. Nitroindazole acts as a selective inhibitor for neuronal nitric oxide synthase, a hemoprotein enzyme that, in neuronal tissue, converts arginine to citrulline and nitric...
.
The subcellular localisation of nNOS in skeletal muscle is mediated by anchoring of nNOS to dystrophin
Dystrophin
Dystrophin is a rod-shaped cytoplasmic protein, and a vital part of a protein complex that connects the cytoskeleton of a muscle fiber to the surrounding extracellular matrix through the cell membrane. This complex is variously known as the costamere or the dystrophin-associated protein complex...
. nNOS contains an additional N-terminal domain, the PDZ domain
PDZ domain
The PDZ domain is a common structural domain of 80-90 amino-acids found in the signaling proteins of bacteria, yeast, plants, viruses and animals...
.
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+. While evidence for ‘baseline’ iNOS expression has been elusive, IRF1IRF1
Interferon regulatory factor 1 is a protein that in humans is encoded by the IRF1 gene.- Function :Interferon regulatory factor 1 was the first member of the interferon regulatory transcription factor family identified...
and NF-κB-dependent activation of the inducible NOS promoter supports an inflammation mediated stimulation of this transcript.
From a functional perspective, it is important to recognize that 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
Reactive nitrogen species
Reactive nitrogen species are a family of antimicrobial molecules derived from nitric oxide and superoxide produced via the enzymatic activity of inducible nitric oxide synthase 2 and NADPH oxidase respectively...
leading to peroxynitrite
Peroxynitrite
Peroxynitrite is the anion with the formula ONOO−. It is an unstable structural isomer of nitrate, NO3−, which has the same formula but a different structure. Although peroxynitrous acid is highly reactive, its conjugate base peroxynitrite is stable in basic solution...
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.
It has been suggested that pathologic generation of nitric oxide
Nitric oxide
Nitric oxide, also known as nitrogen monoxide, is a diatomic molecule with chemical formula NO. It is a free radical and is an important intermediate in the chemical industry...
through increased iNOS production may decrease tubal ciliary beats and smooth muscle contractions and thus affect embryo transport, which may consequently result in ectopic pregnancy
Ectopic pregnancy
An ectopic pregnancy, or eccysis , is a complication of pregnancy in which the embryo implants outside the uterine cavity. With rare exceptions, ectopic pregnancies are not viable. Furthermore, they are dangerous for the parent, since internal haemorrhage is a life threatening complication...
.
eNOS
Endothelial NOS (eNOS), also known as nitric oxide synthase 3 (NOS3), generates NO in blood vesselBlood vessel
The blood vessels are the part of the circulatory system that transports blood throughout the body. There are three major types of blood vessels: the arteries, which carry the blood away from the heart; the capillaries, which enable the actual exchange of water and chemicals between the blood and...
s and is involved with regulating vascular function. A constitutive Ca2+ dependent NOS provides a basal release of NO. eNOS is associated with plasma membranes surrounding cells and the membranes of Golgi bodies within cells. eNOS localisation to endothelial membranes is mediated by cotranslational N-terminal myristoylation
Myristoylation
Myristoylation is an irreversible, co-translational protein modification found in animals, plants, fungi, protozoans and viruses. In this protein modification, a myristoyl group is covalently attached via an amide bond to the alpha-amino group of an N-terminal amino acid of a nascent polypeptide...
and post-translational palmitoylation
Palmitoylation
S-Palmitoylation is the covalent attachment of fatty acids, such as palmitic acid, to cysteine residues of membrane proteins. The precise function of palmitoylation depends on the particular protein being considered. Palmitoylation enhances the hydrophobicity of proteins and contributes to their...
.
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 dismutaseSuperoxide dismutase
Superoxide dismutases are a class of enzymes that catalyze the dismutation of superoxide into oxygen and hydrogen peroxide. As such, they are an important antioxidant defense in nearly all cells exposed to oxygen...
(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.
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 Nω-hydroxy-L-arginine (NOHLA) as an intermediate. 2 mol of O2 and 1.5 mol of NADPH are consumed per mole of NO formed.
Structure
The enzymes exist as homodimers. In eukaryotes, each monomer consisting of two major regions: an N-terminal oxygenaseOxygenase
An oxygenase is any enzyme that oxidizes a substrate by transferring the oxygen from molecular oxygen O2 to it. The oxygenases form a class of oxidoreductases; their EC number is EC 1.13 or EC 1.14....
domain, which belongs to the class of heme-thiolate proteins, and a multi-domain C-terminal reductase
Reductase
-Examples:* 5-alpha reductase* Dihydrofolate reductase* HMG-CoA reductase* Methemoglobin reductase* Ribonucleotide reductase* Thioredoxin reductase* E. coli nitroreductase* Methylenetetrahydrofolate reductase...
, which is homologous to NADPH:cytochrome P450 reductase
Cytochrome P450 reductase
Cytochrome P450 reductase is a membrane-bound enzyme required for electron transfer to cytochrome P450 in the microsome of the eukaryotic cell from a FAD- and FMN-containing enzyme NADPH:cytochrome P450 reductase .-Introduction:...
and other flavoproteins. The FMN binding domain ins 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
Calmodulin
Calmodulin is a calcium-binding protein expressed in all eukaryotic cells...
-binding sequence. The oxygenase domain is a unique extended beta sheet cage with binding sites for heme and pterin.
NOSs can be dimeric
Protein dimer
In biochemistry, a dimer is a macromolecular complex formed by two, usually non-covalently bound, macromolecules like proteins or nucleic acids...
, calmodulin-dependent or calmodulin-containing cytochrome p450-like hemoprotein
Hemoprotein
A hemeprotein , or heme protein, is a metalloprotein containing a heme prosthetic group- an organic compound that allows a protein to carry out a function that it cannot do alone....
that combines reductase and oxygenase catalytic domains in one dimer, bear both flavin adenine dinucleotide (FAD) and flavin mononucleotide
Flavin mononucleotide
Flavin mononucleotide , or riboflavin-5′-phosphate, is a biomolecule produced from riboflavin by the enzyme riboflavin kinase and functions as prosthetic group of various oxidoreductases including NADH dehydrogenase as well as cofactor in biological blue-light photo receptors...
(FMN), and carry out a 5`-electron oxidation of non-aromatic amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
arginine with the aid of tetrahydrobiopterin.
All three isoforms (each of which is presumed to function as a homodimer during activation) share a carboxyl-terminal reductase domain homologous to the cytochrome P450 reductase
Cytochrome P450 reductase
Cytochrome P450 reductase is a membrane-bound enzyme required for electron transfer to cytochrome P450 in the microsome of the eukaryotic cell from a FAD- and FMN-containing enzyme NADPH:cytochrome P450 reductase .-Introduction:...
. They also share an amino-terminal oxygenase domain containing a heme
Heme
A heme or haem is a prosthetic group that consists of an iron atom contained in the center of a large heterocyclic organic ring called a porphyrin. Not all porphyrins contain iron, but a substantial fraction of porphyrin-containing metalloproteins have heme as their prosthetic group; these are...
prosthetic group, which is linked in the middle of the protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
to a calmodulin
Calmodulin
Calmodulin is a calcium-binding protein expressed in all eukaryotic cells...
-binding domain. Binding of calmodulin appears to act as a "molecular switch" to enable electron
Electron
The electron is a subatomic particle with a negative elementary electric charge. It has no known components or substructure; in other words, it is generally thought to be an elementary particle. An electron has a mass that is approximately 1/1836 that of the proton...
flow from flavin prosthetic groups in the reductase domain to heme. This facilitates the conversion of O2 and L-arginine to NO
Nitric oxide
Nitric oxide, also known as nitrogen monoxide, is a diatomic molecule with chemical formula NO. It is a free radical and is an important intermediate in the chemical industry...
and L-citrulline. The oxygenase domain of each NOS isoform also contains an BH4 prosthetic group, which is required for the efficient generation of NO. Unlike other enzymes where BH4 is used as a source of reducing equivalents and is recycled by dihydrobiopterin reductase
Dihydrobiopterin reductase
Dihydrobiopterin is a compound produced in the synthesis of dopamine, norepinephrine and epinephrine through production of the intermediate 3,4-dihydroxy phenylalanine, also known as dopa...
, BH4 activates heme-bound O2 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 "Ca2+ sensitive" but it is now known that they are present in many different cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
types and that expression is regulated under specific physiological conditions.
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
Heme
A heme or haem is a prosthetic group that consists of an iron atom contained in the center of a large heterocyclic organic ring called a porphyrin. Not all porphyrins contain iron, but a substantial fraction of porphyrin-containing metalloproteins have heme as their prosthetic group; these are...
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
Negative feedback
Negative feedback occurs when the output of a system acts to oppose changes to the input of the system, with the result that the changes are attenuated. If the overall feedback of the system is negative, then the system will tend to be stable.- Overview :...
regulatory role on NOS3, and therefore vascular endothelial cell function. 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. 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. 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
Enzyme induction and inhibition
Enzyme induction is a process in which a molecule induces the expression of an enzyme.Enzyme inhibition can refer to* the inhibition of the expression of the enzyme by another molecule...
of NOS2.
External links
- The Nobel Prize in Physiology or Medicine 1998
- University of Edinburgh, School of Chemistry - NO Synthase
- Nitric Oxide Synthase in Proteopedia