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IntroductionNitric oxide (NO) is an important
signaling biomolecule and a cytotoxin.
NO is produced by nitric oxide synthase (NOS)◦Neuronal (nNOS)
Neuronal tissues; NO as a neurotransmitter
◦Inducible (iNOS) Macrophages; NO as a cytotoxin
◦Endothelial (eNOS) Endothelial cells; NO as a vasodilator
ReactionCatalizes a 2-step reaction
from L-Arg to L-citrulline and NO
Two major domains:◦ Catalytic (oxygenase) domain:
Heme L-Arginine Tetrahydrobiopterin (H4B)
◦ Reductase Domain NADPH FAD, FMN
Step 1 of Mechanism
Conversion of L-Arg to Nw-hydroxy-L-arginine (NHA)
Similar to cytochrome P450
H4B
NADPH
Heme Binding
Cys-415 binds to heme Fe
Tyr-706 binds to heme propionate oxygen
Trp-409 can p-stack with heme ring
L-Arginine/NHA BindingGlu-592 binds in
three placesPro-565
H-bonds to a H2O, which then binds to the guanidinium N of L-arg.
Hydroxylated N-H of NHA binds to O2 oxygen bound to heme Fe
Zinc Tetrathiolate ClusterNOS is only active as a homodimer; the
monomeric form is inactiveThe dimer is held together by a Zn2+ ion
complexed to 2 pairs of Cys residues from each paired monomer
High [NO] can cause S-nitrosation of 2 Cys residues, which leads to release of Zn and formation of inactive NOS monomers
Sequence Alignment
Key:1. Alpha Helices2. Beta Sheets3. Heme Binding Residues4. L-Arg/NHA Binding Residues5. H4B Binding Residues6. Zn Tetrathiolate Cys Residues
Kinetic Data
A. Rate of NO production of WT rat nNOS monitored by Hb assay at room temperature. B. Lineweaver-Burk plot for dtermination of Km and Vmax.
References (Structure Paper) Doukov, T., Li, H., Soltis, M., and Poulos, T. L. (2009)
Single crystal structure and absorption spectral characterizations of nitric oxide synthase complexed with Nw-hydroxy-L-arginine and diatomic ligands. Biochemistry 48, 10246-10254.
Kerwin, J. R. Jr., Lancaster, J. R. Jr., and Feldman, P. L. (1995) Nitric oxide: a new paradigm for second messengers. J. Med. Chem. 38, 4343-4362.
Mitchell, D. A., Erwin, P. A., Michel, T., and Marletta, M. A. (2005) S-Nitrosation and regulation of inducible nitric oxide synthase. Biochemistry 44, 4636-4647.
Raman, C. S., Li, H., Martasek, P., Kral, 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.
Li, D., Kabir, M., Stuehr, D. J., Rousseau, D. L, and Yeh, S. R. (2007) Substrate- and isoform-specific dioxygen complexes of nitric oxide synthase. J. Am. Chem. Soc. 129, 6943-6951.
Fang, J., Ji, H., Lawton, G. R., Xue, F., Roman, L. J., and Silverman, R. B. (2009) L337H Mutant of rat neuronal nitric oxide synthase resembles human neuronal nitric oxide synthase toward inhibitors. J. Med. Chem. 52, 4533-4537
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