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 Endothelial (eNOS) Endothelial cells; NO as a vasodilator

Reaction 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 Conversion of L- Arg to Nw- hydroxy-L-arginine (NHA) Similar to cytochrome P450 NADPH H4B

Step 2 of Mechanism Conversion 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 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

H4B Binding Carbonyl H-Bonds Arg-596 Heme propionate group Ser-334 Trp-678 Phe-691 Arg-596 Heme propionate group H4B

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 Key: Alpha Helices Beta Sheets Heme Binding Residues L-Arg/NHA Binding Residues H4B Binding Residues 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