1. Nitric Oxide Synthase Joey Klen

2. 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

3. 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

4. Step 1 of Mechanism
Conversion of L- Arg to Nw- hydroxy-L-arginine (NHA)
Similar to cytochrome P450
NADPH
H4B

5. Step 2 of Mechanism Conversion of NHA to L-citrulline and NO
Not like P450
H4B
NADPH

6. Heme Binding Cys-415 binds to heme Fe
Tyr-706 binds to heme propionate oxygen
Trp-409 can p-stack with heme ring

7. L-Arginine/NHA Binding
Glu-592 binds in three places
Pro 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

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

9. 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

10. Sequence Alignment Key: Alpha Helices Beta Sheets
Heme Binding Residues
L-Arg/NHA Binding Residues
H4B Binding Residues
Zn Tetrathiolate Cys Residues

11. Alignment Continued

12. 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.

13. 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,
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,
Mitchell, D. A., Erwin, P. A., Michel, T., and Marletta, M. A. (2005) S-Nitrosation and regulation of inducible nitric oxide synthase. Biochemistry 44,
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,
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,
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,