Nitric Oxide SynthaseJoey Klen

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

Step 2 of MechanismConversion

of NHA to L-citrulline and NO

Not like 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

H4B BindingCarbonyl H-

Bonds◦ Ser-334◦ Trp-678◦ Phe-691

Arg-596Heme propionate

group

H4B

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

Alignment Continued

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