Nitric Oxide Synthase
Nitric Oxide Synthase (NOS) is an enzyme catalysing the formation of L-Citrulline and Nitric Oxide[1] (NO) from L-arginine. NOS is a homodimeric protein with 125- to 160-kD subunits.
In mammals three isozymes of NOS has been identified: Neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS)[1]. (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 or induced systems in the organism. 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.
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 two cysteines from each oxygenase domain. Binding of the domains is caused by calmodulin (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, 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 H4B (5,6,7,8-tetrahydrobiopterin)and the already mentioned Heme ion (Fe(III)). 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 reaction of NOSThe 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:
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.
The Oxygenase domain of NOSThe Oxygenase domain of NOS
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The contains the active site of the enzyme. The active site binds the substrate (Arginine) which is converted into citruline and NO (explained in details below). The domain has three cofactors bound:
((6R).5,6,7,8-Tetrahydrobiopterin)
(Heme)
General StructureGeneral Structure
The oxygenase domain can be divided into three subdomains. Firstly, the substrate-binding subdomain, which is crescent in shape, binds the substrate in an interior pocket of the crescent. The Heme group is located between the tips of the crescent shape, thus closing the pocket in which the substrate is located. Secondly the H4B binding subdomain serves as a cap for the cavity created by the substrate binding domains crescent shape. Thirdly there is a subdomain with two helical bundles making up a hydrophobic core, however this subdomain is not thought to take part in the catalytic activity of the enzyme.
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 (L-arginine) in the distal pocket by hydrogen bindings both to the guanidino[2] end and the amino acid end. is shown in green with the heme and H4B shown. NOS binds its substrate by coordinating CO(or O2) to the heme at the site occupied by oxygen[2](it is the opposite site of the Cys coordination to heme - look in the 'heme' section). The binding of substrate leads to a 2-step transformation first to N-hydroxy-L-arginine (the tightly bound intermediate) and then NO and L-Citrulline. The product NO can then either diffuse out of the or bind to the heme and function in NO auto-inhibition though this inhibition is diverse throughout the 3 isoforms[3].
PDB structures used in the section above: [3NOS], [2G6H]
H4BH4B
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is a cofactor. NOS contains two molecules of , one in each monomer. The active center forms is a part of the cavity already described. This cavity can be visualized as a . Here H4B helps substrate interactions by lining the active-center tunnel and hydrogen bonding to the heme propionate amd to alfa7. Amd and alfa7 are two elements involved in L-Arg binding. This gives H4B the opportunity to play an important role in the control of subunit interactions and active-center 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[4].
The H4B is bound by hydrogen-bonds to several of the molecules surrounding it, including the substrate L-Arg. The substrate is H-bonded to the 4-keto group of pterin, and to one of the heme propionate groups ( the heme propionate group has two carboxylate oxygens in use for H-bonds). These oxygens are further H-bonded to the 4-keto group of pterin, through water, and directly to N(3) and NH2 on C (2)[4]. The overall picture of all the H-bonds can be seen by clicking on the figure on the left.
But H4B is not only a structurel cofactor, it also plays a very important role in NO synthesis, donating an electron to the heme.[5] 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 FeIIO2 intermediate to be reduced to oxidants that are able to react with Arg and N-hydroxy-L-arginine (NOHA) [5] If H4B was not present the FeIIO2 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[5].
PDB structures used in the section above: [3NOS], [2G6H]
HemeHeme
Heme is a prosthetic group containing an iron atom in the center of a large organic ring called porphyrin(See picture). The heme group can have several functions: it can act as a transporter of diatomic gases, as a chemical catalyst, detect diatomic gases, and take part in electron transfer. In NOS the heme both binds a diatomic molecule (CO, NO, O2) and functions in the multi electron pathway [6].
As mentioned, the heme group takes part in the creation of the cavity running through the monomer. Arginine/Citrulline diffuses in and out this cavity. The heme group in the cavity is held in place by van der Waals interactions with hydrophobic and aliphatic side chains of the protein making up the cavity. The propionic acid groups of heme forms several hydrogen bonds with water molecules inside the cavity. One of these groups hydrogenbonds to H4B. The iron in heme in pentacoordinated[7], with its axial ligand supplied by a specific Cys S atom.
ZincZinc
In order for Nos to be active it has to dimerize and bind H4B. The two monomers are held together by a single structural
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which is situated at the interface of the dimer[7]. The zinc ion is tetrahedrally coordinated and has four cysteins bound as ligands (two from each monomer - Cys109 and Cys104). Further, it is found that zinc binds together the oxygenase domains of the monomers. The zinc ion is found at a region which connects the N-terminal hook and the subunit core. The coordination of zinc arranges the N-terminal hooks so that they interact with their own subunit. However, when there is no zinc ion present, two of the thiolate ligands (cysteines) form a disulfide bond connecting the two subunits[8].
PDB structures used in the section above: [2G6H]
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, here is only shown one subunit of the domain:
(Nicotinamide adenine dinucleotide phosphate)
The reductase domain is, as mentioned, bound to an oxygenase domain by a calmodulin linkerCalmodulin. This linker responds to Ca2+ -ions (constitutive NOS isoforms). The calmodulin linker consists of 32 residues and contains a binding region for the Ca2+ -ions. This binding is found to be crucial in that it induces a conformational change which is essential for 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, thus the linker acts as a hinge. The electron transfer occurs two times per NO molecule produced. The first transfer supplies an electron for the conversion of L-Arginine to its intermediate, the second transfer for the conversion of the intermediate 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 [9]. The Iron atom in the heme group is reduces from iron (III) to iron (II) which catalyses the substrate reaction.
Structures used in the section above: [1TLL]
ReferencesReferences
- ↑ 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
- ↑ Fan B, Wang J, Stuehr DJ, Rousseau DL. NO synthase isozymes have distinct substrate binding sites. Biochemistry. 1997 Oct 21;36(42):12660-5. PMID:9376373 doi:http://dx.doi.org/10.1021/bi9715369
- ↑ Rousseau DL, Li D, Couture M, Yeh SR. Ligand-protein interactions in nitric oxide synthase. J Inorg Biochem. 2005 Jan;99(1):306-23. PMID:15598509 doi:http://dx.doi.org/10.1016/j.jinorgbio.2004.11.007
- ↑ 4.0 4.1 Raman CS, Li H, Martasek P, Kral V, Masters BS, Poulos TL. Crystal structure of constitutive endothelial nitric oxide synthase: a paradigm for pterin function involving a novel metal center. Cell. 1998 Dec 23;95(7):939-50. PMID:9875848
- ↑ 5.0 5.1 5.2 Wei CC, Wang ZQ, Meade AL, McDonald JF, Stuehr DJ. Why do nitric oxide synthases use tetrahydrobiopterin? J Inorg Biochem. 2002 Sep 20;91(4):618-24. PMID:12237227
- ↑ Li H, Igarashi J, Jamal J, Yang W, Poulos TL. Structural studies of constitutive nitric oxide synthases with diatomic ligands bound. J Biol Inorg Chem. 2006 Sep;11(6):753-68. Epub 2006 Jun 28. PMID:16804678 doi:http://dx.doi.org/10.1007/s00775-006-0123-8
- ↑ 7.0 7.1 Fischmann TO, Hruza A, Niu XD, Fossetta JD, Lunn CA, Dolphin E, Prongay AJ, Reichert P, Lundell DJ, Narula SK, Weber PC. Structural characterization of nitric oxide synthase isoforms reveals striking active-site conservation. Nat Struct Biol. 1999 Mar;6(3):233-42. PMID:10074942 doi:http://dx.doi.org/10.1038/6675
- ↑ Crane BR, Rosenfeld RJ, Arvai AS, Ghosh DK, Ghosh S, Tainer JA, Stuehr DJ, Getzoff ED. N-terminal domain swapping and metal ion binding in nitric oxide synthase dimerization. EMBO J. 1999 Nov 15;18(22):6271-81. PMID:10562539 doi:http://dx.doi.org/10.1093/emboj/18.22.6271
- ↑ Garcin ED, Bruns CM, Lloyd SJ, Hosfield DJ, Tiso M, Gachhui R, Stuehr DJ, Tainer JA, Getzoff ED. Structural basis for isozyme-specific regulation of electron transfer in nitric-oxide synthase. J Biol Chem. 2004 Sep 3;279(36):37918-27. Epub 2004 Jun 17. PMID:15208315 doi:http://dx.doi.org/10.1074/jbc.M406204200