Because NO cannot be stored, released, or inactivated by conventional regulatory mechanisms, biosynthetic regulation is more important for NO than for other mediators. Indeed, NO synthase (NOS), the NO biosynthetic enzyme, is one of the most regulated enzymes in biology. Initial efforts to purify the enzyme were unsuccessful because of a rapid loss of enzyme activity on purification. Observations that calmodulin is required for NOS activity in the brain led to a simple purification of neuronal NOS (nNOS) to homogeneity.43 Using this approach, other groups purified nNOS,44,45 inducible NOS46– 49 (iNOS), and endothelial NOS (eNOS).50 Molecular cloning of the cDNA for neuronal,51,52 endothelial,53– 55 and inducible56– 59 forms of NOS has helped elucidate NOS function. The structure of NOS reveals numerous regulatory mechanisms.
NOS oxidizes the guanidine group of L-arginine in a process that consumes five electrons and results in the formation of NO with stoichiometric formation of L-citrulline. L-N ω-substituted arginine analogues such as L-Nω-nitroarginine (L-NNA) and L-N ω-methylarginine (L-NMA), function as NOS inhibitors.60 The inhibition of NOS by these substrate analogues can initially be reversed by simultaneous application of excess arginine consistent with their competitive blockade of the active site. However, following prolonged exposure NOS is irreversibly inhibited by some of these agents. The irreversible inactivation of the macrophage enzyme and brain enzymes by L-NMA requires simultaneous incubation with NOS cofactors suggesting “mechanism-based” inhibition.61,62 The time-dependent inactivation of the brain enzyme by L-NNA63 is independent of NOS enzymatic turnover.64
NOS isoforms display modest differences in their sensitivity to various arginine analogs. L-NNA is a more potent inhibitor of the brain and endothelial enzymes (Ki = 200–500 nM), and L-Nω-aminoarginine (L-NAA) is a more potent blocker of the inducible enzyme (Ki = 1–5 μM). Clinically useful inhibitors of NOS will likely need to be isoform specific. Intensive work by numerous pharmaceutical companies has focused on discovery of such selective antagonists. High throughput drug screening yielded a highly selective nNOS antagonist65 that appears useful for mitigating neuronal injury in an animal model of cerebral ischemia (see below).
Oxidative enzymes generally employ redox active cofactors. NOS is unprecedented in employing five. The cloning of NOS's oxidative enzymes (discussed below) indicates a close homology of the C-terminal half of NOS with cytochrome P450 reductase (CPR) including consensus sequences for NADPH, FAD, and FMN binding.51 While NADPH is a stoichiometric substrate, the two flavins copurify with NOS in a ratio of 1 eq each of FAD and FMN per NOS monomer.46,66 FAD slowly dissociates from NOS and must be exogenously supplied for maximal activity. The close homology of NOS with CPR suggests that electrons follow the same path through NOS as they do through CPR; that is, NADPH initially reduces FAD, which, in turn, reduces FMN. In fact, NOS and CPR share a domain thought to be involved in this electron transfer.51
CPR supplies the reducing equivalents from NADPH to the heme-containing cytochrome P450 enzymes. This mechanism is apparently shared by NOS isoforms, which contain 1 eq of iron-protoporphyrin IX per NOS monomer.67– 69 Furthermore, NOS displays reduced CO difference spectra typical of cytochrome P450 with wavelength absorbance maximum at 445 nM, which provided that first indication of a heme-binding cysteinyl ligand. CO inhibits purified NOS, which is also consistent with the participation of a cytochrome P450-type heme in the reaction. NO itself appears to exert feedback inhibition of NOS,70– 72 perhaps by interacting with the enzyme's heme prosthetic group. Optical-difference spectroscopy indicates that heme binds to the substrate arginine prior to participating in the oxygenation reactions.73 Heme-substrate binding is also the initial event in catalysis with the various cytochrome P450s. Unlike other mammalian cytochrome P450 enzymes, NOSs are unique because they are soluble and their flavin (reductase) and heme (oxygenase)-containing domains are fused in a single polypeptide. A bacterial fatty acid monooxygenase, P450BM3, also has been identified as a soluble, self-contained P450 system.74
NOS is also regulated by tetrahydrobiopterin (H4B). While isolated iNOS and eNOS are absolutely dependent on H4B,50,75,76 purified nNOS retained substantial activity in the absence of added H4B.43,77 This discrepancy is explained by the tight binding of H4B to nNOS such that H4B copurifies with the enzyme.78 It was initially assumed that H4B functions directly in the hydroxylation of arginine by analogy to its role in aromatic amino acid hydroxylase enzymes. Kaufman and coworkers79 challenged this notion in experiments, proposing that H4B stabilizes NOS. Their conclusion was based on experiments with nNOS that showed that H4B functions catalytically, is not recycled, and does not affect the initial rate of NOS. Marletta and colleagues,80 based on experiments with pterin analogues used to probe the iNOS reaction, also suggested that H4B stabilizes NOS.
In many cell types, the availability of H4B is rate limiting for NO biosynthesis. Resting macrophages, smooth muscle cells, and hepatocytes contain only minute amounts of H4B. However, following stimulation with cytokines, cellular H4B levels dramatically increase to support NO synthesis.81,82 The mechanism for this increase is that immunostimulants selectively induce expression of GTP cyclohydrolase I, the first and rate-limiting enzyme for the synthesis of H4B. This increase in GTP cyclohydrolase I occurs at the transcriptional level and follows a time course that closely parallels the induction of iNOS transcription.81,82 These findings suggest that pterin synthesis inhibitors may offer a strategy for antagonizing iNOS activity.
The recent 3D structural characterization of the N-terminal half of iNOS by x-ray crystallography determines, at atomic resolution, the location of cofactors in the oxygenase domain.83,84 This work reveals an unusual fold and heme environment in NOS that resembles a baseball catcher's mitt, and which helps stabilize activated oxygen intermediates that are key for catalysis. Confirming mutagenesis work, the heme is axially coordinated to proximal cysteine-194. In the dimeric crystal, arginine is bound to glutamate-371 and stacks with heme in a hydrophobic pocket near the dimer interface. The iNOS dimer structure supports previous proposals that H4B plays a primary role in regulating NOS dimerization and active site formation, but that H4B does not appear to play a direct redox role in catalysis.83
The conversion of arginine to NO is catalyzed in two independent steps (Fig. 168-3). The first step is a two-electron oxidation of arginine to Nω-hydroxyarginine (NHA).85 Although this hydroxylated intermediate is tightly bound to NOS, under certain conditions NHA can be isolated as a product.64 This hydroxylation step resembles a classical P450 type monooxygenation reaction utilizing 1 eq of NADPH and 1 eq of O2.85 The hydroxylation reaction is accelerated by H4B, requires calcium and calmodulin as activators, and is blocked by CO.64,68,85
Mechanism of NO synthesis. NOS catalyzes a five-electron oxidation of a guanidine nitrogen of L-arginine to generate NO and L-citrulline. L-Hydroxyarginine is formed as an intermediate that is tightly bound to the enzyme. Both steps in the reaction are dependent on calcium and calmodulin and are enhanced by tetrahydrobiopterin.
The steps in the pathway from NHA to NO and citrulline are less clear. Any proposed mechanism should account for experiments which find that this oxidation (a) utilizes 0.5 eq NADPH, (b) requires O2 and calcium/calmodulin, (c) is accelerated by H4B, and (d) is inhibited by CO and arginine analogues with a pharmacology similar to that seen in the initial hydroxylation reaction.64,68,85 In one model consistent with these data, NOS would use both its reductase and heme domains for successive independent oxidations of arginine at a common active site with heme directly functioning in the activation of molecular oxygen. For the first hydroxylation, both reducing equivalents for oxygen activation derive from NADPH. It has been speculated that NHA and NADPH each provide one electron for the second oxidation step.86 This explains both the 0.5 stoichiometry of NADPH utilization and accounts for the unusual five-electron chemistry of NO biosynthesis. The crystallographic structure of dimeric iNOS oxygenase domain is consistent with this proposed mechanism. Indeed, these ligand-bound NOS crystal structures suggest that the different protonation state of arginine vs. N-hydroxyarginine differentiates the two chemical steps of NO synthesis.83
Molecular Cloning of NOS Isoforms
Isolation of the brain isoform43 permitted its molecular cloning.51 The cDNA predicts a polypeptide of 160 kD and was striking in having 36 percent identity to CPR in its C-terminal half, the NOS reductase domain, which contains the binding sites of NADPH, FAD, and FMN (Fig. 168-4). This homology to CPR is shared by all NOSs cloned to date, and reflects the oxidative mechanism of NO biosynthesis. The sequence of the N-terminal half of NOSs, the heme domains, is not similar to any cloned gene. Although the classic P450 heme-binding cysteinyl peptide sequence is absent, the amino acids surrounding cysteine-414 showed some of the expected homology. Indeed, mutagenesis and x-ray crystallographic studies have definitively demonstrated cysteine-414 of nNOS as the site for heme coordination.
Alignment of the co-factor recognition sites within NOS enzymes. Predicted sites for binding heme, calmodulin (CaM), FMN, FAD, and NADPH are noted. The N-terminal PDZ domain in nNOS, and the myristoylation (Myr) and palmitoylation (Palm) sites in eNOS are marked. The unique alternatively spliced region in nNOSμ from skeletal muscle corresponds to the domain not present in iNOS.
The reductase domain of NOS shares many functional properties with CPR. nNOS catalyzes a rapid NADPH-dependent reduction of cytochrome c. In the absence of arginine, NOS can transfer electrons from NADPH to O2
− and form O2 − and H2O2.87 The formation of these reactive oxygen intermediates may contribute to glutamate neurotoxicity and neurodegeneration as discussed below. Near the middle of the nNOS cDNA there is an amphipathic alpha helix domain, which conforms to the consensus sequence for calmodulin binding.51 This assignment was confirmed by experiments showing that peptides corresponding to this region bind calmodulin with low nanomolar affinity in a calcium-dependent manner.88 Cloning of nNOS from human cerebellum predicts 94 percent acid identity with the rat protein.89
Genomic cloning has shown that nNOS is a huge and complex gene that occupies at least 160 kb90 and maps to chromosome position 12q24.2.91,92 The most common mRNA isolated from brain derives from 29 exons, but several alternatively spliced forms of nNOS mRNA in brain have been described. Esumi and coworkers93 reported the first example of alternative splicing of nNOS. These investigators found that, in mouse cerebellum and in human neuroblastoma cell lines, a small percentage of nNOS mRNA has a 315-nucleotide deletion corresponding to nucleotides 1510–1824. Structural analysis of the corresponding part of the nNOS gene indicated that the deletion corresponds to exons 9 and 10. The greatest diversity of nNOS transcripts occurs in the 5′ untranslated region. Molecular cloning of nNOS from human brain cDNA identified two distinct 5′-terminal exons that are spliced to a common exon 2. Genomic cloning demonstrated that the unique exons occur within 300 bp of each other. Transcriptional analyses revealed that expression of the 5′-terminal exons is regulated by separate upstream promoter regions.94 Independent analysis by Marsden et al. indicates that at least eight different examples of exon 1 of nNOS are expressed in human tissues.95 Again, the diverse 5′ ends are spliced into a common exon 2. Despite this immense diversity of 5′ mRNA variants of nNOS, most transcripts encode the same 155-kD protein. What might be the biologic advantage of such a system? Clearly, this complexity could allow for tissue- and stage-specific gene expression. It is also possible that the multiple nNOS mRNAs have different functional properties. The distinct 5′ termini may either influence mRNA translational efficiency or alter mRNA localization or stability.
eNOS was cloned independently by three labs using low stringency screening strategies based on the DNA sequence of nNOS.53– 55 Overall, the predicted sequence shares 60 percent identity with nNOS. Consensus binding sites for FAD, FMN, NADPH, and calmodulin are conserved between the brain and endothelial isoforms. A unique feature of the eNOS gene is a consensus sequence for N-terminal myristoylation. [3H]Myristate is directly incorporated into eNOS, and mutation of the myristoylation sequence renders eNOS soluble.96,97 Insertion of the myristoyl group into the plasma membrane presumably accounts for the enzyme's particulate location.
Near its N-terminus, the eNOS protein also contains an unusual glycine/leucine penta-repeat that is flanked on both sides by cysteine residues. These cysteines are sites of palmitoylation, which is another posttranslational fatty acid modification of the enzyme.98,99 Palmitoylation of eNOS refines the subcellular target of the enzyme and helps recruit the protein both to the Golgi complex and to caveolae, small cave-like invaginations of the plasma membrane.100,101 Palmitoylation of eNOS appears to be a dynamic process that contributes to enzyme activation and recycling following agonist stimulation.102
Human eNOS is a large gene that contains 25 exons spanning 21 kb on the 7q35-36 region of chromosome number 7, the same chromosome that contains CPR.91,103 Characterization of 5′-flanking genomic regions indicates that the endothelial NO synthase promoter is “TATA-less” and contains proximal promoter elements that are commonly found in constitutively expressed endothelial transcripts, including Sp1 and GATA elements.91 Functional analysis shows that the canonical Sp1 site is required for basal promoter activity.104 The 5′ promoter region of the human gene contains AP-1, AP-2, NF-1, heavy metal, acute-phase response shear stress, and sterol-regulatory elements.91 This complex array of 5′ sequence motifs fit with work showing eNOS expression is regulated by diverse physiological stimuli including shear stress,105 physical exercise,106 hypoxia,107,108 estrogen treatment,109 and low levels of oxidized low-density lipoproteins.110,111
Cloning of iNOS from rodent macrophages was independently achieved by three labs. Two groups used nNOS cDNA as a homologous probe,56,58 while the third used an iNOS antibody for expression cloning.57 Overall, the amino acid sequence is 50 percent identical to nNOS and 51 percent identical to eNOS. The iNOS cDNA predicts a protein of 133 kD with consensus binding sequences for NADPH, FAD, FMN, and calmodulin.
An inducible calcium-independent NOS activity is well characterized in human hepatocytes following treatment with LPS, γ-interferon, tumor necrosis factor-α, and interleukin-1β.112 Cloning of this cDNA indicated 82 percent identity with mouse iNOS, suggesting that it represents the human-inducible isoform.59 Independently, a nearly identical human-inducible NOS gene was cloned from articular chondrocytes activated with interleukin-1β.113 The human iNOS gene maps to chromosome 17qcen-q12.114– 116
For inducible NOS, one would expect the regulatory region of the gene to determine the rate of synthesis of enzyme protein. Initial characterization of the promoter region of the gene for iNOS revealed a pattern for complex regulation.117 There appear to be two distinct regulatory regions upstream of the TATA box, which is 30 base pairs upstream of the transcription start site. One of these, region 1, lies 50 to 200 base pairs upstream of the start site. Region 1 contains LPS-related response elements, such as the binding site for NF-IL6 and the κB binding site for NF-κB,118 indicating that this region regulates the LPS induced expression of iNOS. Region 2, which is 900 to 1000 base pairs upstream of the start site, does not itself directly regulate NOS expression, but provides a tenfold increase above the 75-fold increase in NOS expression provided by region 1. Region 2 contains motifs for interferon regulatory factor-1 and is responsible for interferon-γ-mediated regulation.119,120 In sum, LPS and interferon-γ responsive elements occur in two distinct regulatory genes with LPS directly stimulating iNOS expression while interferon-γ acts only in the presence of LPS.
This unique organization of gene enhancers may explain important aspects of inflammation. In sepsis, LPS is released from Gram-negative bacterial cell walls and circulates throughout the body to stimulate inflammatory responses. By contrast, interferon-γ is released locally and serves to augment inflammatory responses in specific cell populations close to its release. LPS alone stimulates macrophages to a limited extent. Interferon-γ elaborated by infiltrating lymphocytes can prime the macrophages for a maximal response to LPS. Thus, maximal production of NO is restricted to those cells needed to kill the invader, thereby minimizing damage to adjacent tissue.
Similar to iNOS in rodents, human iNOS is regulated in a complex manner by specific cytokine combinations. Characterization of the proximal human iNOS promoter region demonstrates major differences in sequence and potential cis-acting elements as compared to the rodent iNOS promoter.114 At a functional level, the human iNOS promoter also contrasts markedly from mouse iNOS. Whereas the proximal 1 kb of the murine iNOS promoter mediates responses to cytokines, reporter constructs containing the first 4 kb upstream of human iNOS show no cytokine-inducible activity.121,122 Instead, cytokine-responsive regions, containing functional NF-κB sites,123 occur 5 to 10 kb upstream of the transcriptional start site.124 This absence of inducible promoter elements near the transcription start site is very unusual for a cytokine-regulated gene and helps explain the unique regulation of iNOS by diverse inflammatory stimuli.