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Nitric Oxide Synthase - NOSNitric Oxide Synthase - NOS

Nitric Oxide Synthase (NOS) is a group of enzymes catalysing L-arginine to yield L-Citrulline and Nitric Oxide[1] (NO). NOS is a homodimeric protein with 125- to 160-kD subunits. An overview of the NOS homodimer is given below. All cofactors are included and the electron transfer pathway which takes place in NOS is indicated.

File:NOS HOMODIMER.JPG

The NOS homodimer is composed of two types of domains: an oxygenase domain and a reductase domain. Each subunit is held together by a Zinc ion, which is bound by the amino acid Cystein present in the oxygenase. Binding of the domains is caused by CaM. The reductase domain supplies electrons for the NOS reaction which takes place in the oxygenase domain. The reductase domain contains two redox-active prosthetic groups, FAD and FMN. NADPH binds to the domain and passes on an electron to FAD which passes the electron on to FMN. FMN is a Flavin mononucleotide. FMN passes the electron on to the Heme in the oxygenase domain on the opposite subunit. The oxygenase domain contains BH₄ (5,6,7,8-tetrahydrobiopterin)and the already mentioned Heme ion (Fe(III)). These two are also redox active groups. BH₄ is required by NOS in order to produce NO and not H₂O₂. Besides Heme and BH₄, the oxygenase domain binds the substrate L-arginine which takes part in the NO synthase reaction (see below).

In mammals three isozymes of NOS has been identified: Neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). (~The NOS enzymes is found in numeral organisms. Most facts used here is from the human NOS, but sites from different organisms are used.~). Neuronal NOS is producing NO in the nervous tissue in both the peripheral and the central nervous system. nNOS is functioning in cell signaling and communication - a vital part of the nervous tissue. Inducible NOS is connected with the immune system or in general...(!?). Endothelial NOS is controlling the amount of NO signaling in the endothelial cells eg. blood vessel dilation.

The NOS reaction requires five redox-active cofactors.

Substrate bindingSubstrate binding

The active site is highly conserved in the different NOS species. Thus it is possible to discuss substrate binding i general terms. The NOS enzyme binds its substrate by hydrogen bindings both to the guanidino[2] end and the amino acid end (EVT FIG!).

spinqualitylabelsall models Substrate binding Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

binds its substrate (L-arginine) by coordinating CO to the heme at the site occupied by oxygen.....

The reaction of NOS:The reaction of NOS:

As previously described NOS is an enzyme split in to different domains; the N-terminal oxygenase domain and the C-terminal reductase domain.

The oxygenase domain is where the production of NO takes place, whereas the reductase domain provides the electrons necessary to drive the reaction in the oxygenase domain. The reaction is:

INDSÆT FIGUR

The amino acid L-Arginine is turned in to L-Citrulline and NO. The reaction is driven by the oxidation of NADPH to NADP+, which in total yields 5 electrons for the reaction. The reaction above therefore takes both the oxygenase and the reductase domain into account.

StructureStructure

NOS is a homodimeric protein with 125- to 160-kD subunits. An overview of the NOS homodimer is given below. All cofactors are included and the electron transfer pathway which takes place in NOS is indicated.

File:NOS HOMODIMER.JPG

The NOS homodimer is composed of two types of domains: a oxygenase domain and a reductase domain. Each subunit is held together by a Zinc ion, which is bound by the amino acid Cystein present in the oxygenase. Binding of the domains is caused by CaM. The reductase domain supplies electrons for the NOS reaction which takes place in the oxygenase domain. The reductase domain contains two redox-active prothetic groups, FAD and FMN. NADPH binds to the domain and passes on an electron to FAD which passes the electron on to FMN. FMN is a Flavin mononucleotide. FMN passes the electron on to the Heme in the oxygenase domain on the opposite subunit. The oxygenase domain contains BH4 (5,6,7,8-tetrahydrobiopterin)and the already mentioned Heme ion (Fe(III)). These two are also redox active groups.BH4 is required by NOS in order to produce NO and not H2O2. Besides Heme and BH4, the oxygenase domain binds the substrate L-arginine which takes part in the NO synthase reaction (see below).

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H4BH4B

is a cofactor. NOS contains two molecules of H₄B, one in each monomer. The active center forms a kind of tunnel, because of the dimeric structure. This gives H4B the opportunity to play a big role in the control of subunit interactions and active-center formation. H₄B therefor is more of a structurel cofactor, in that it keeps the dimer stabilized by integration in to the hydrophobic parts of the dimer. Here it helps substrate interactions by lining the active-center channel and hydrogen bonding to the heme propionate amd to alfa7 which is two elements involved in L-Arg binding. So H₄B is not the molecule that hydroxylates the substrate (L-Arg) nor activating the hemebound oxygen. Pterin induces some changes in the heme invironment, including ordering of the active -center channel, increased sequestration (sequestration (om proces) the action of forming a chelate or other stable compound with an ion or atom or molecule so that it is no longer available for reactions) of the heme ligand Cys194, and extension of the negative hemeA propionate away from the distal heme pocket may account for the 50mV increase in heme redox potential and low-high spin shift of the ferric heme iron in the presence og H₄B. It also may increase the oxygen activation, because of the pterin-induced 70-fold increase in autoxidation of the ferrous heme-dioxy complex. Fra artiklen: structure of NOS oxygenase dimer with pterin and substrate)))

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Template:STRUCTURE 2g6h

The Oxygenase domain of NOSThe Oxygenase domain of NOS

((6R).5,6,7,8-Tetrahydrobiopterin)

(Arginine)

(Heme)

The Reductase Domain of NOSThe Reductase Domain of NOS

The reductase domain of the NOS homodimer will not be discussed thoroughly at this page. However a short discussion of the electron transfer which occurs will be given along with an introduction to the bound cofactors and the general structure.

The has three cofactors bound:

(Nicotinamide adenine dinucleotide phosphate)

(Flavin adenine dinucleotide)

(Flavin mononucleotide)

The reductase domain is, as mentioned, bound to an oxygenase domain by a calmodulin linker. This linker responds to Ca2+ -ions (constitutive NOS isoforms). The calmodulin linker is consists of 32 residues and contains a binding region for the Ca2+-ions. This binding is found to be crucial it induces a conformational change which is essential for the electron transfer. It is important to emphasize that the electron transfer occurs from the reductase domain of one subunit to the oxygenase domain of the opposite subunit (i.e. a trans transfer). The conformational change induced by Ca2+-ions brings the mentioned reductase and oxygenase domains closer together, therefore the linker acts like a hinge. The electron transfer occurs two times per produced NO molecule, first electrons are passed on for the conversion of L-Arginine to its intermediate, secondly for the conversion of the intermediate to produce Citruline and NO. In general the reductase domain can be divided into three binding domains: the NADPH binding domain, the FAD binding domain, and the FMN binding domain. The NADPH and FAD binding domains are associated whereas the FAD and FMN domains are connected by an α-helical binding domain. An electron is donated by NADPH, which passes the electron on to FAD. FAD shuttles on the electron to FMN. The FMN binding domain is a flexible domain and here the conformational change occurs, the Calmodulin linker rotates the reductase domain and oxygenase domain along a vertical axis, thus bringing the reductase domain closer to the opposite oxygenase domain. The electron can then due to shorter distance be passed on the the Heme group bound by the oxygenase domain.