Nitric Oxide in Biology
The study of Nitric Oxide (NO) leapt to life in the late 1980s when it was discovered to act as a signalling mediator in an array of physiological processes, including regulation of blood flow/pressure, neurotransmission and immune response. This breakthrough led to the 1998 Nobel prize for physiology being awarded to R. Furchgott, L. Ignarro and F. Murad. The public's imagination was also aroused by the prominent role of NO in the signalling pathway targeted by the blockbuster drug Viagra. 

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NOS active site

The NO synthases generate NO from L-arginine when stimulated as part of a signalling pathway or as a response of the immune system. In order to maintain rigorous control of NO production they have evolved a number of different regulatory mechanisms. These mechanisms are being investigated on a molecular level with the aid of structural models, molecular biological and biophysical techniques. 

The NO synthases are composed of two globular protein modules (domains) connected via a flexible protein strand. The oxygenase domain consists of a unique catalytic centre responsible for NO production, while the reductase domain supplies the electrons required for NO synthesis by catalysing NADPH dehydrogenation and transferring the electrons generated across the interface between the two domains. This electron transfer is activated by the binding of the small clam-like protein, calmodulin, to a sequence specific binding site on the flexible hinge. For the neuronal and endothelial NO synthase isoforms calmodulin only binds at increased concentrations of calcium, thereby forming a calcium dependent molecular switch. This is one of several known NO synthase regulation mechanisms.

The different components of NO synthase are illustrated in the figure above. The reductase domain (shown binding two flavin molecules) is linked via the helical peptide in the centre to the oxygenase domain, which is dimeric. The small protein calmodulin binds Ca²⁺ ions and then activates NO synthase by complexing with the interdomain peptide link. Although the structure of the oxygenase dimer is known and the structure of the reductase can be considered to be similar to that of P450 reductase (as used above), the structure of fully assembled NO synthase and the mechanism by which calmodulin activates the enzyme are still under investigation. Some of our recent work has defined an autoinhibitory loop in the reductase domain of nNOS which, together with a C-terminal extension, appears to form part of a conformational locking mechanism. We now have evidence that the lock is closed by NADPH binding, which causes the FMN to be shielded from its electron transfer partner. The lock is released by calmodulin binding, which results in enzyme activation.

“Redox Properties of the Isolated FMN- and FAD-binding Domains of Neuronal NO Synthase”. Garnaud, P.E., Koetsier, M., Ost, T.W.B. and Daff, S. (2004) Biochemistry 43, 11035-11044.

“An appraisal of multiple NADPH binding-site models proposed for P450 BM3, Cytochrome P450 reductase and NO synthase.” Daff, S. (2004) Biochemistry 43, 3929-3932.

“Heme-thiolate proteins: Cytochrome P450 versus Nitric Oxide Synthase, Power versus Control” Chapman, S.K., Daff, S. and Ost, T.W.B. (2003) The Biochemist 25, 20-23.

“Calmodulin-dependent regulation of Mammalian NO synthase” Daff, S. (2003) Biochem. Soc. Trans.31, 502-505.

“Calmodulin Activates Electron Transfer Through Neuronal NO Synthase Reductase Domain By Releasing an NADPH-Dependent Conformational Lock”. Craig, D.H., Chapman, S.K., and Daff, S. (2002) J. Biol. Chem. 277, 33987-33994.

"Electron transfer in nitric-oxide synthase" Sagami, I., Sato, Y., Noguchi, T., Miyajima, M., Rozhkova, E., Daff, S. and Shimizu, T. (2002) Coord. Chem. Rev. 226, 179-186.

“Interactions between the Isolated Oxygenase and Reductase Domains of Neuronal Nitric-oxide Synthase. ASSESSING THE ROLE OF CALMODULIN”. Rozhkova, E.A., Fujimoto, N., Sagami, I., Daff, S.N., and Shimizu, T. (2002) J. Biol. Chem. 277, 16888-16894.

“Rapid Calmodulin-Dependent Interdomain Electron Transfer in Neuronal Nitric-Oxide Synthase Measured by Pulse Radiolysis”. Kobayashi, K., Tagawa, S., Daff, S., Sagami, I., and Shimizu, T. (2001) J. Biol. Chem. 276, 39864-39871.

“Intrasubunit and Intersubunit Electron Transfer in Neuronal Nitric-oxide Synthase: Effect of Calmodulin on Heterodimer Catalysis”. Sagami, I., Daff, S., and Shimizu, T. (2001) J. Biol. Chem. 276, 30036-30042.

"Control of Electron Transfer in Neuronal NO Synthase" Daff, S., Noble,M. A., Craig, D., Rivers, S. L., Chapman, S. K., Munro, A. W., Fujiwara,S., Rozhkova, E., Sagami, I., & Shimizu, T. (2001) Biochem. Soc. Trans. 29, 147-152.

"Important Role of Tetrahydrobiopterin in NO Complex formation and Interdomain Electron Transfer in Neuronal Nitric Oxide Synthase" Noguchi, T., Sagami,I., Daff, S., & Shimizu, T. (2001) Biochem. Biophys. Res. Commun.282, 1092-1097.

"Roles of the Heme Proximal Side Residues Tryptophan 409 and Tryptophan421 of Neuronal Nitric Oxide Synthase in the Electron Transfer Reaction" Yumoto, T., Sagami, I., Daff, S. & Shimizu, T. (2000) J. Inorg.Biochem. 82, 163-170.

Aromatic Residues and Neighboring Arg414 in the (6R)-5,6,7,8-Tetrahydro-L-Biopterin Binding Site of Full-length Neuronal Nitric-oxide Synthase Are Crucial in Catalysis and Heme Reduction with NADPH
Sagami, I., Sato, Y., Daff, S., & Shimizu, T. (2000) J. Biol. Chem. 275, 26150-26157.

Potentiometric Analysis of the Flavin Cofactors of Neuronal Nitric Oxide Synthase.
 Noble, M.A., Munro, A.W., Rivers, S.L., Robledo, L., Daff, S.N., Yellowlees, L.J., Shimizu, T., Sagami, I., Guillemette, J.G. & Chapman, S.K. (1999) Biochemistry 38, 16413-16418.

The 42-Amino Acid Insert in the FMN Domain of Neuronal Nitric Oxide Synthase Exerts Control over Ca²⁺/Calmodulin-dependent Electron Transfer.
Daff, S., Sagami, I., & Shimizu, T. (1999) J. Biol. Chem. 274, 30589-30595.

Crucial Role of Lys423 in the Electron Transfer of Neuronal Nitric Oxide Synthase
Shimanuki, T., Sato, H., Daff, S., Sagami, I., & Shimizu, T. (1999) J. Biol. Chem. 274, 26956-26961.

Autoxidation Rates of Neuronal Nitric Oxide Synthase: Effects of the Substrates, Inhibitors, and Modulators.
Sato, H., Sagami, I., Daff, S. & Shimizu, T. (1998) Biochem. Biophys. Res. Comm. 253, 845-849.

Chiral Recognition at the Heme Active Site of Nitric Oxide Synthase Is Markedly Enhanced by L-Arginine and 5, 6, 7, 8-Tetrahydrobiopterin.
Nakano, K., Sagami, I., Daff, S. & Shimizu, T. (1998) Biochem. Biophys. Res. Comm. 248, 767-772.

CO binding studies of nitric oxide synthase: effects of the substrate, inhibitors and tetrahydrobiopterin.
Sato, H., Nomura, S., Sagami, I., Ito, O., Daff, S. & Shimizu, T. (1998) FEBS Lett. 430, 377-380.

Heme: the most versatile redox centre in biology.
Chapman, S.K., Daff, S. & Munro, A.W. (1997) Structure and bonding 88, 39-70.