Nitric Oxide Synthase: Difference between revisions

=Introduction=

In mammals three isozymes of NOS have been identified: Neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS)<ref>Flemming, I., Chapter 3, Biology of Nitric Oxide Synthases, from Microcirculation, Editors Ronald F. Tuma, R. F., Durán, W. N., and Ley, K., 2nd edition, Academic Press, 2008</ref>. (The NOS enzymes are found in numeral organisms. Most facts used here are 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. Endothelial NOS is controlling the amount of NO signaling in the endothelial cells eg. blood vessel dilation.  An overview of the structural organization of the NOS homodimer is given below. All cofactors are included and the electron transfer pathway which takes place in NOS is indicated.  

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Each NOS monomer is composed of two domains: an N-terminal oxygenase domain and a C-terminal reductase domain. Each subunit is held together by a zinc ion, which is bound by two cysteines from each oxygenase domain. Calmodulin binds to a linker region between the two domains and is necessary for activity. 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, flavin adenine dinucleotide (FAD) and Flavin mononucleotide (FMN). Nicotinamide adenine dinucleotide phosphate(NADPH) binds to the domain and passes on an electron to FAD which passes the electron on to FMN. FMN passes the electron on to the heme in the oxygenase domain on the opposite subunit. The oxygenase domain contains 5,6,7,8-tetrahydrobiopterin (H₄B) and the already mentioned heme. These two are also redox active groups. Besides Heme and H₄B, the oxygenase domain binds the substrate L-arginine which takes part in the NO synthase reaction (see below).

The NO production takes plase in the oxygenase domain, whereas the reductase domain provides the electrons necessary to drive the reaction in the oxygenase domain.

The reaction is:

The reaction occurs in two steps. In the first step L-arginine is turned in to N-hydroxy-arginine. In the second step L-citrulline and NO are formed. The reaction utilizes 2O₂ and 3 electrons from 3/2 NADPH.<ref>PMID:17014963</ref>

===H₄B===

[[image:bh4.png|left|frame|Structure of tetrahydrobiopterin]]

<scene name='Sandbox_5/Nos_oxygenase_bh4/11'>H4B</scene> is an essential cofactor in NOS and in the [[aromatic amino acid hydroxylases]]. NOS contains two molecules of <scene name='Sandbox_5/Begge_h4b/1'>H4B</scene>, one in each monomer. The active site forms part of the cavity already described. This cavity can be visualized as a <scene name='Nitric_oxide_synthase/Substratebinding_distal_pocket/2'>tunnel</scene>. Here H₄B helps substrate interactions by lining the active-center tunnel and hydrogen bonding to the heme propionate and to alfa helix 7. The propionate group and alpha helix 7 are also involved in the L-Arg binding. This gives H₄B the opportunity to play an important role in the control of subunit interactions and active site formation. H₄B is therefore more or less a structural cofactor and has a stabilizing effect. Its structural importance is further reconned to play a role in dimer formation (dimerization requires bound zinc ion along with H₄B), and major conformational changes leading to the formation of the active site channelform<ref name="Raman">PMID:9875848</ref>.

[[image:H4b_hydrogenbindinger2.png|thumb|left|Hydrogenbondings in H₄B binding site ([[2nse]]) ]]

But H₄B is not only a structural cofactor, it also plays a very important role in NO synthesis, donating an electron to the heme.<ref name="heme">PMID:12237227</ref> H₄B can deliver an electron to the heme much faster than the reductase domain can, therefor H₄B is used by NOS in the Arg hydroxylation, activating O₂ by providing the second electron. Thus, H₄B is a kinetically prefered electron donor. As shown in the reaction (bottom right, click for enlargement) the second electron, that H₄B donates, helps the Fe^IIO₂ intermediate to be reduced to oxidants that are able to react with Arg and N-hydroxy-L-arginine (NOHA) <ref name="heme"/> If H₄B was not present the Fe^IIO₂ intermediate would decay to superoxide and ferric enzyme due to the reductase domain being slower to deliver an electron than the proces of decay is to happen. H₄B is faster than both of these processes<ref name="heme"/>.

PDB structures used in the section above: [[3nos]], [[2g6h]]

Structures used in the section above: [[1tll]]

==References==

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