• Prosthetic group features
• Nitric oxide synthase (NOS) reaction
• NOS isozymes
• Cofactor binding sites in the three NOS isozymes
• Electron flow in NOS
• NOS in enzyme databases
• NOS in motif databases
• NOS in alignment databases
• NOS in 3­D databases
• References

	Prosthetic group			Oxidation states
FAD	Flavin adenine dinucleotide (FAD)			FAD; FAD· (semiquinone); FADH2
FMN	Flavin mononucleotide (FMN)			FMN; FMN· (semiquinone); FMNH2
BH4	(6R)­5,6,7,8­Tetrahydrobiopterin (BH4)			BH4 (active form); BH2 (inactive form)
Zn	Zn(SCys)4			[Zn(Cys)4]2-
Haem	Haem type	Haem iron coordination	Axial iron ligand(s)	Formal iron oxidation/spin states
	Haem b	Pentacoordinate	SCys	FeII (S=2); FeIII (S=5/2)
		Hexacoordinate	SCys; NO, CO or other ligand	FeII (S=0); FeIII (S=1/2)
		Hexacoordinate	SCys; O (O·)	FeIV (S=1)

Nitrogen monoxide (·NO), commonly referred to as nitric oxide in the biochemical literature [1], is a free radical generated in biological systems. ·NO functions at low concentrations as a signal in many diverse physiological processes such as blood pressure control, neurotransmission, learning and memory, and at high concentrations as a defensive cytotoxin (see list of reviews on structure and function of nitric oxide synthases). Nitric oxide synthase (NOS; EC 1.14.13.39) enzymes produce ·NO by catalysing a five­electron oxidation of a guanidino nitrogen of L­arginine (L­Arg). Oxidation of L­Arg to L­citrulline occurs via two successive monooxygenation reactions producing N­hydroxy­L­arginine as an intermediate (1). 2 mol of O₂ and 1.5 mol of NADPH are consumed per mole of ·NO formed [2 and references therein].

	NADPH O2		½NADPH O2		+	·N=O	(1)
L­Arginine		N­hydroxy­L­arginine		L­Citrulline

iNOS and nNOS are soluble and found predominantly in the cytosol, while eNOS is membrane associated. eNOS localisation to endothelial membranes is mediated by cotranslational N­terminal myristoylation and post­translational palmitoylation. The enzymes exist as homodimers, each monomer consisting of two major domains: N­terminal oxygenase domain, which belongs to the class of haem-thiolate proteins, and C­terminal reductase domain, which is homologous to NADPH:P450 reductase. The interdomain linker between the oxygenase and reductase domains contains a CaM­binding sequence. nNOS contains an additional N­terminal domain (`DHR domain') which shows homology to syntrophins, a family of dystrophin­binding proteins, some protein tyrosine phosphatases, protein kinases and some other proteins [4]. The subcellular localisation of nNOS in skeletal muscle is mediated by anchoring of nNOS to dystrophin. A schematic alignment of cofactor binding sites of the three NOS isozymes follows:

nNOS

DHR

haem

CaM

FMN

FAD

NADPH

iNOS

haem

CaM

FMN

FAD

NADPH

eNOS

Myr

haem

CaM

FMN

FAD

NADPH

All NOS isozymes are catalytically self­sufficient. The electron flow in the ·NO synthase reaction is

						CaM
NOS	NADPH		FAD		FMN		haem		O2	(1.1)

The 3­D structures have been reported for iNOS oxygenase domain (iNOS_ox) complexes with the NOS inhibitors imidazole and aminoguanidine [8], iNOS_ox dimer complex with BH₄ and L­Arg [9], and BH₄­bound and BH₄­free dimers of constitutive eNOS oxygenase domain (eNOS_ox) [10]. NOS_ox has a novel nonmodular/ß fold, resembling "a baseball catcher's mitt for a left hand" [8]. The fold consists of a winged ß­sheet with projecting ß­hairpins and flanking ­helices. Haem is situated in the ß­sheet `palm' with its distal face directed toward a large cavity. In iNOS_ox-imidazole complex, the low­spin haem iron is axially coordinated to proximal Cys­194 and to exogenous imidazole. The L­Arg analogue aminoguanidine binds in the distal pocket adjacent to the haem is such a way that one guanidino nitrogen is directed toward the activated oxygen binding site.

The crystal structure of eNOS_ox dimer reveals a zinc ion tetrahedrally coordinated to pairs of symmetry­related Cys residues at the dimer interface [10].

NOS in enzyme databases

ENZYME	LIGAND	BRENDA	Official name	Alternative names
1.14.13.39	1.14.13.39	1.14.13.39	Nitric oxide synthase	NO synthase; NO synthetase; NOS

NOS in motif databases

PRINTS ID	PRINTS AC	PROSITE/BLOCKS ID	PROSITE AC	BLOCKS AC
FPNCR	PR00371	-	-	-
FLAVODOXIN	PR00369	FLAVODOXIN	PS00201	BL00201

NOS in alignment databases

Protein Superfamily	Protein Homology Domain	Pfam	LPFC 3­D alignment
03633; nitric oxide synthase	00062; flavodoxin	PF00175; oxidored_fad PF00258; flavodoxin	-

PDB	scop	BSM	RELI Base	Header	¹
1noc	-	1noc	-	Inducible nitric oxide synthase oxygenase domain (114) (complex with type I E. coli chloramphenicol acetyl transferase and imidazole); mouse macrophage (recombinant form expressed in Escherichia coli)	-
1nos	-	1nos	-	Inducible nitric oxide synthase oxygenase domain (114) (complex with imidazole); mouse macrophage (recombinant form expressed in Escherichia coli)	-
2nos	-	2nos	-	Inducible nitric oxide synthase oxygenase domain (114) (complex with aminoguanidine and imidazole); mouse macrophage (recombinant form expressed in Escherichia coli)	-

¹ Macromolecular Structures abstract. Full text is available to BioMedNet Members

References

1. Koppenol, W.H. and Traynham, J.G. (1996) Say NO to nitric oxide: Nomenclature for nitrogen­ and oxygen­containing compounds. Methods Enzymol. 268, 3-7.
2. Liu, Q. and Gross, S.S. (1996) Binding sites of nitric oxide synthases. Methods Enzymol. 268, 311-324.
3. Knowles, R.G. and Moncada, S. (1994) Nitric oxide synthases in mammals. Biochem. J. 298, 249-258.
4. Ponting, C.P. and Phillips, C. (1995) DHR domains in syntrophins, neuronal NO synthases and other intracellular proteins. Trends Biochem. Sci. 20, 102-103.
5. Giovanelli, J., Campos, K.L. and Kaufman, S. (1991) Tetrahydrobiopterin, a cofactor for rat cerebellar nitric oxide synthase, does not function as a reactant in the oxygenation of arginine. Proc. Natl. Acad. Sci. USA 88, 7091-7095.
6. Hevel, J.M. and Marletta, M.A. (1992) Macrophage nitric oxide synthase: relationship between enzyme­bound tetrahydrobiopterin and synthase activity. Biochemistry 31, 7160-7165.
7. Presta, A., Siddhanta, U., Wu, C., Sennequier, N., Huang, L., Abu­Soud, H.M., Erzurum, S. and Stuehr, D.J. (1998) Comparative functioning of dihydro­ and tetrahydropterins in supporting electron transfer, catalysis, and subunit dimerization in inducible nitric oxide synthase. Biochemistry 37, 298-310.
8. Crane, B.R., Arvai, A.S., Gachhui, R., Wu, C., Ghosh, D.K., Getzoff, E.D., Stuehr, D.J. and Tainer, J.A. (1997) The structure of nitric oxide synthase oxygenase domain and inhibitor complexes. Science 278, 425-431.
9. Crane, B.R., Arvai, A.S., Ghosh, D.K., Wu, C., Getzoff, E.D., Stuehr, D.J. and Tainer, J.A. (1998) Structure of nitric oxide synthase oxygenase dimer with pterin and substrate. Science 279, 2121-2126.
10. Raman, C.S., Li, H., Martásek, P., Král, V., Masters, B.S.S. and Poulos, T.L. (1998) Crystal structure of constitutive endothelial nitric oxide synthase: a paradigm for pterin function involving a novel metal center. Cell 95, 939-950.

Bibliography on structural studies of nitric oxide synthases

Reviews on nitric oxide synthases

The Nitric Oxide Home Page

Directory of P450­containing Systems