Nitric oxide (NO) is a small, gaseous, uncharged molecule produced by the nitric oxide synthase (NOS) family of enzymes, which convert l-arginine and O2 to l-citrulline and NO. NO possesses a relatively long half life and diffuses readily through cellular membranes, allowing it to serve a variety of unique roles as a neurotransmitter, primarily by stimulating the enzyme soluble guanylyl cyclase (sGC) to produce cyclic GMP. NO synthesis is coupled to Ca2+ influx by the Ca2+-activated signaling molecule Calmodulin (CaM), which binds NOS directly. In mammals, NO has been implicated in the regulation of systemic vasomotor tone and blood pressure, bronchial and gastrointestinal smooth muscle relaxation, penile erection, inflammatory responses, and leukocyte-mediated microbe destruction, as well as neuromodulatory effects on behavior, memory, and nociception1.
Three distinct isoforms of the NOS protein are found in mammals: endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS) [Diagram] [Reset]. All NOS isoforms are dimers of two identical subunits, with the continuous polypeptide chain of a single nNOS subunit being 1429 amino acids in length and possessing a mass of approximately 160 kDa. Each NOS subunit contains three conserved domains, the reductase domain, the calmodulin binding domain, and the oxygenase domain2 [Diagram] [Reset]. The CaM binding domain (residues 720-743) binds Ca2+-activated CaM, allowing NOS to respond to changes in intracellular calcium levels. The reductase domain (residues 743-1429) binds three ligands, nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and flavin mononucleotide (FMN), and serves to transfer electrons from NADPH to the oxygenase heme-binding domain. Using NADPH-derived electrons, the oxygenase domain (residues 1-720) converts O2 and l-arginine to NO and l-citrulline. The protein at left is the reductase domain of nNOS (nNOSred) isolated from rat cortical tissue.
Amino acids 1232-1396 constitute the NADPH binding domain <>. NADPH binds at one end of a five-stranded parallel β-sheet <>. Gln-1324 hydrogen bonds to the adenosine moiety of NADPH, which also undergoes hydrophobic stacking interactions with Tyr-1322 <>. A network of ionic interactions anchors the NADPH in the binding site: the 2' phosphate interacts with Ser-1313, Arg-1314, and Arg-1400 <>, located on the CTN. Arg-1010, from the FAD-binding domain, and Arg-1284 contributing additional ionic interactions <>.
The FMN binding domain consists of amino acids 750-942, with amino acids 943-989 comprising the connecting domain (CD) and 1059-1078 comprising a beta-finger <>. These domains are necessary for rotational flexibility of the FMN-binding domain (see below). The FMN-binding domain consists of a five-stranded parallel β-sheet flanked by five α-helices <>. When FMN binds, Gly-810 accepts a Hydrogen-bond from the FMN N5 atom, and the ring plane of FMN is stacked between the rings of Phe-809 and Tyr-889 residues <>.
The flavin-containing face of the FMN-binding domain inserts into the cup-shaped depression formed by the continuous unit of the FAD, NADPH, and connecting domains (the FNR/CD unit), burying a total surface of 2,500 Å <>. A double salt bridge between residues Glu-816 and Arg-1229 directly links the FMN and FAD domains <>, along with an extensive network of hydrophobic contacts, H-bonds, and salt bridges3 <>. The CD aligns the alloxazine rings of the bound flavins to facilitate electron transfer from FAD to FMN: in this conformation, the xylene ring methyl groups of the flavins are only 4.8 Å apart <>.
In this FMN/FAD interface, the bound FMN is completely buried and cannot transfer electrons to the oxygenase domain—rotational flexibility of the FMN-binding domain is necessary for this transfer to occur. It is theorized that the flexible hinge region <> at the C-terminus of the FMN-binding domain serves as a pivot point, allowing the domain to rotate so that the bound FMN is approximately 15 Å from the heme region of the trans oxygenase domain across the dimer, close enough for electron transfer to proceed. This requirement for rotation of the FMN-binding domain explains why transfer of electrons to the heme is the rate limiting step in NO synthesis. The gap in the hinge structure results from weakly defined electron density, which reflects the flexible nature of this region.
An autoinhibitory insert composed of 45 amino acids (residues 827-872) is located within the FMN-binding domain <>. This insert contains an α-helix anchored within a hydrophobic pocket between the FMN- and NADPH-binding domains. The autoinhibitory helix (AH) also contains a conserved motif of three hydrophobic residues, Tyr-841, Phe-845, and Val-848, preceded by two basic residues, Arg-838 and Lys-839 < >, which has been termed the “AH motif”3. This pattern resembles canonical Ca2+/CaM-binding motifs.
AH most likely regulates NO synthesis by inhibiting the production of NO at low Ca2+ concentrations in one of two theoretical ways. First, AH could competitively bind Ca2+/CaM, preventing activated CaM from contacting the CaM-binding domain when it is present only at low concentrations. Second, the interactions of AH with the FMN- and NADPH-binding domains could contribute to a locked position in which the FMN domain is unable to rotate. In this case, high concentrations of Ca2+ would induce CaM binding and subsequent release of the FMN domain to participate in electron transfer. Whichever of these theoretical mechanisms is correct, it is expected that the ultimate function of AH is to inhibit NO production under low Ca2+ concentrations.
The C-terminal tail (CT) of nNOSred, attached to the terminus of the NADPH-binding domain, also serves to repress NOS activity when CaM activation is low. CT can be divided into two regions. The C-terminal segment (CTC, residues 1414-1429), which is variable in length and sequence among NOS isozymes, is not included in this protein structure. The N-terminal segment (CTN, residues 1397-1413) <>, on the other hand, is highly conserved. CTN contains an α-helical segment that sits across a negatively charged groove at the FAD/FMN interface <>. Residues 1397-1400, at the base of the helix, are attached via hydrogen bonds to Tyr-889 and Asp-1351 of the FMN- and NADPH-binding domains, respectively <>. Arg-1400 on the CTN also binds to the 2’ phosphate of NADPH, as noted above. These interactions lock the FMN-binding domain in the electron-accepting position, inhibiting NO synthesis. Activated CaM binding to the Ca2+/CaM domain disrupts these interactions, allowing the FMN domain to rotate freely and NO synthesis to occur.
A regulatory phosphorylation site, Ser-1412 < >, is also found on CTN at the end of the a-helix. The addition of a negatively charged phosphate causes the helix to be repelled from the negative groove in which it lies, displacing the CTN from its normal position and releasing the FMN-binding domain. Phosphorylation of Ser-1412 therefore serves to relieve the repression of NOS activity.
These two regulatory mechanisms in the reductase subunit ensure that NO sythesis by NOS is tightly coupled to Ca2+ influx, an essential control for the overall biological activity of the NOS protein.
1Rawlingson A (2003). Nitric oxide, inflammation and acture burn injury. Burns. 29: 631-640.
2Li H & Poulos TL (2005). Structure-function studies on nitric oxide synthases. J. Inorg. Biochem. 99: 293-305.
3Garcin ED, Bruns CM, Lloyd SJ, Hosfield DJ, Tiso M, Gachhui R, et al. (2004). Structural Basis for Isozyme-specific Regulation of Electron Transfer in Nitric Oxide Synthase. J. Biol. Chem. 279: 37918-37927.