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Nitric Oxide Synthase Joey Klen. Introduction. Nitric 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

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  • Nitric 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


  • Catalizes 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
Step 1 of Mechanism

  • Conversion of L-Arg to Nw-hydroxy-L-arginine (NHA)

  • Similar to cytochrome P450



Step 2 of mechanism
Step 2 of Mechanism

  • Conversion of NHA to

    L-citrulline and NO

  • Not like P450



Heme binding
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 binding
L-Arginine/NHA Binding

  • Glu-592 binds in three places

  • Pro-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

H 4 b binding
H4B Binding

  • Carbonyl H-Bonds

    • Ser-334

    • Trp-678

    • Phe-691

  • Arg-596

  • Heme propionate group


Zinc tetrathiolate cluster
Zinc Tetrathiolate Cluster

  • NOS is only active as a homodimer; the monomeric form is inactive

  • The 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
Sequence Alignment


Alpha Helices

Beta Sheets

Heme Binding Residues

L-Arg/NHA Binding Residues

H4B Binding Residues

Zn Tetrathiolate Cys Residues

Kinetic data
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.


  • (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