The function of the small insertion in the hinge subdomain in the control of constitutive mammalian nitric-oxide synthases

Control of nitric oxide (NO) synthesis in the constitutive nitric-oxide synthases (NOS) by calcium/calmodulin is exerted through the regulation of electron transfer from NADPH through the reductase domains. This process has been shown previously to involve the calmodulin binding site, the autoinhibitory insertion in the FMN binding domain, and the C-terminal tail. Smaller sequence elements also appear to correlate with control. Although some of these elements appear well positioned to function in control, they are poorly conserved; their role in control is neither well established nor defined by available information. In this study mutations have been induced in the small insertion of the hinge subdomain, which has been shown recently to form a beta hairpin in structural studies of the neuronal NOS reductase domains adjacent to the calmodulin site and the autoinhibitory element. Modification of the small insertion in neuronal NOS tends to increase cytochrome c reduction but not NO synthetic activity; some modifications or deletions in the corresponding region in endothelial NOS modestly increase activity under some conditions. Unexpectedly, some minor changes in the sequence introduce a loss in the content of heme relative to flavin cofactors. Taken together, these results suggest that the small insertion protects the calmodulin binding site and that it may be a modulator of NOS activity.     

The Role of Formin Tails in Actin Nucleation,Processive Elongation,and Filament Bundling

Formins are multidomain proteins that assemble actin in a wide variety of biological processes. They both nucleate and remain processively associated with growing filaments, in some cases accelerating filament growth. The well conserved formin homology 1 and 2 domains were originally thought to be solely responsible for these activities. Recently a role in nucleation was identified for the Diaphanous autoinhibitory domain (DAD), which is C-terminal to the formin homology 2 domain. The C-terminal tail of the Drosophila formin Cappuccino (Capu) is conserved among FMN formins but distinct from other formins. It does not have a DAD domain. Nevertheless, we find that Capu-tail plays a role in filament nucleation similar to that described for mDia1 and other formins. Building on this, replacement of Capu-tail with DADs from other formins tunes nucleation activity. Capu-tail has low-affinity interactions with both actin monomers and filaments. Removal of the tail reduces actin filament binding and bundling. Furthermore, when the tail is removed, we find that processivity is compromised. Despite decreased processivity, the elongation rate of filaments is unchanged. Again, replacement of Capu-tail with DADs from other formins tunes the processive association with the barbed end, indicating that this is a general role for formin tails. Our data show a role for the Capu-tail domain in assembling the actin cytoskeleton, largely mediated by electrostatic interactions. Because of its multifunctionality, the formin tail is a candidate for regulation by other proteins during cytoskeletal rearrangements.     

Exploring the electron transfer properties of neuronal nitric-oxide synthase by reversal of the FMN redox potential

In nitric-oxide synthase (NOS) the FMN can exist as the fully oxidized (ox), the one-electron reduced semiquinone (sq), or the two-electron fully reduced hydroquinone (hq). In NOS and microsomal cytochrome P450 reductase the sq/hq redox potential is lower than that of the ox/sq couple, and hence it is the hq form of FMN that delivers electrons to the heme. Like NOS, cytochrome P450BM3 has the FAD/FMN reductase fused to the C-terminal end of the heme domain, but in P450BM3 the ox/sq and sq/hq redox couples are reversed, so it is the sq that transfers electrons to the heme. This difference is due to an extra Gly residue found in the FMN binding loop in NOS compared with P450BM3. We have deleted residue Gly-810 from the FMN binding loop in neuronal NOS (nNOS) to give Delta G810 so that the shorter binding loop mimics that in cytochrome P450BM3. As expected, the ox/sq redox potential now is lower than the sq/hq couple. Delta G810 exhibits lower NO synthase activity but normal levels of cytochrome c reductase activity. However, unlike the wild-type enzyme, the cytochrome c reductase activity of Delta G810 is insensitive to calmodulin binding. In addition, calmodulin binding to Delta G810 does not result in a large increase in FMN fluorescence as in wild-type nNOS. These results indicate that the FMN domain in Delta G810 is locked in a unique conformation that is no longer sensitive to calmodulin binding and resembles the "on" output state of the calmodulin-bound wild-type nNOS with respect to the cytochrome c reduction activity.     

FMN fluorescence in inducible NOS constructs reveals a series of conformational states involved in the reductase catalytic cycle

Nitric oxide synthases (NOSs) produce NO as a molecular signal in the nervous and cardiovascular systems and as a cytotoxin in the immune response. NO production in the constitutive isoforms is controlled by calmodulin regulation of electron transfer. In the tethered shuttle model for NOS reductase function, the FMN domain moves between NADPH dehydrogenase and oxygenase catalytic centers. Crystal structures of neuronal NOS reductase domain and homologs correspond to an 'input state', with FMN in close contact with FAD. We recently produced two domain 'output state' (oxyFMN) constructs showing calmodulin dependent FMN domain association with the oxygenase domain. FMN fluorescence is sensitive to enzyme conformation and calmodulin binding. The inducible NOS (iNOS) oxyFMN construct is more fluorescent than iNOS holoenzyme. The difference in steady state fluorescence is rationalized by the observation of a series of characteristic states in the two constructs, which we assign to FMN in different environments. OxyFMN and holoenzyme share open conformations with an average lifetime of ~4.3 ns. The majority state in holoenzyme has a short lifetime of ~90 ps, probably because of FAD-FMN interactions. In oxyFMN about 25-30% of the FMN is in a state with a lifetime of 0.9 ns, which we attribute to quenching by heme in the output state. Occupancy of the output state together with our previous kinetic results yields a heme edge to FMN distance estimate of 12-15 ?. These results indicate that FMN fluorescence is a valuable tool to study conformational states involved in the NOS reductase catalytic cycle.     

Removal of a putative inhibitory element reduces the calcium-dependent calmodulin activation of neuronal nitric-oxide synthase

Neuronal nitric-oxide synthase (NOS) and endothelial NOS are constitutive NOS isoforms that are activated by binding calmodulin in response to elevated intracellular calcium. In contrast, the inducible NOS isoform binds calmodulin at low basal levels of calcium in resting cells. Primary sequence comparisons show that each constitutive NOS isozyme contains a polypeptide segment within its reductase domain, which is absent in the inducible NOS enzyme. To study a possible link between the presence of these additional polypeptide segments in constitutive NOS enzymes and their calcium-dependent calmodulin activation, three deletion mutants were created. The putative inhibitory insert was removed from the FMN binding regions of the neuronal NOS holoenzyme and from two truncated neuronal NOS reductase enzymes in which the calmodulin binding region was either included or deleted. All three mutant enzymes showed reduced incorporation of FMN and required reconstitution with exogenous FMN for activity. The combined removal of both the calmodulin binding domain and the putative inhibitory insert did not result in a calmodulin-independent neuronal NOS reductase. Thus, although the putative inhibitory element has an effect on the calcium-dependent calmodulin activation of neuronal NOS, it does not have the properties of the typical autoinhibitory domain found in calmodulin-activated enzymes.     

Nitric oxide synthase regulatory sites. Phosphorylation by cyclic AMP-dependent protein kinase, protein kinase C, and calcium/calmodulin protein kinase; identification of flavin and calmodulin binding sites.

Nitric oxide (NO) is an important molecular messenger accounting for endothelial-derived relaxing activity in blood vessels, mediating cytotoxic actions of macrophages, and functioning as a neurotransmitter in the brain and periphery. NO synthase (NOS) from brain has been purified to homogeneity and molecularly cloned. We now report that NOS is stoichiometrically phosphorylated by cAMP dependent protein kinase, protein kinase C, and calcium/calmodulin-dependent protein kinase, with each kinase phosphorylating a different serine site on NOS. Activation of PKC in transfected cells reduces NOS enzyme activity by approximately 77% in intact cells and by 50% in protein homogenates from these cells. Utilizing fluorescence spectroscopy we find that purified monomer NOS contains 1 molar equivalent of both FMN and FAD. This stoichiometry is supported by enzymatic digestion of the flavins with phosphodiesterase, and titration of the FMN with a specific FMN binding protein. We demonstrate that purified NOS is labeled by a photoaffinity derivative of calmodulin. These recognition sites on NOS provide multiple means for regulation of NO levels and "cross-talk" between second messenger systems.     

Identification of Redox Partners and Development of a Novel Chimeric Bacterial Nitric Oxide Synthase for Structure Activity Analyses

Production of nitric oxide (NO) by nitric oxide synthase (NOS) requires electrons to reduce the heme iron for substrate oxidation. Both FAD and FMN flavin groups mediate the transfer of NADPH derived electrons to NOS. Unlike mammalian NOS that contain both FAD and FMN binding domains within a single polypeptide chain, bacterial NOS is only composed of an oxygenase domain and must rely on separate redox partners for electron transfer and subsequent activity. Here, we report on the native redox partners for Bacillus subtilis NOS (bsNOS) and a novel chimera that promotes bsNOS activity. By identifying and characterizing native redox partners, we were also able to establish a robust enzyme assay for measuring bsNOS activity and inhibition. This assay was used to evaluate a series of established NOS inhibitors. Using the new assay for screening small molecules led to the identification of several potent inhibitors for which bsNOS-inhibitor crystal structures were determined. In addition to characterizing potent bsNOS inhibitors, substrate binding was also analyzed using isothermal titration calorimetry giving the first detailed thermodynamic analysis of substrate binding to NOS.     

Chimeric enzymes of cytochrome P450 oxidoreductase and neuronal nitric-oxide synthase reductase domain reveal structural and functional differences

The nitric-oxide synthases (NOSs) are comprised of an oxygenase domain and a reductase domain bisected by a calmodulin (CaM) binding region. The NOS reductase domains share approximately 60% sequence similarity with the cytochrome P450 oxidoreductase (CYPOR), which transfers electrons to microsomal cytochromes P450. The crystal structure of the neuronal NOS (nNOS) connecting/FAD binding subdomains reveals that the structure of the nNOS-connecting subdomain diverges from that of CYPOR, implying different alignments of the flavins in the two enzymes. We created a series of chimeric enzymes between nNOS and CYPOR in which the FMN binding and the connecting/FAD binding subdomains are swapped. A chimera consisting of the nNOS heme domain and FMN binding subdomain and the CYPOR FAD binding subdomain catalyzed significantly increased rates of cytochrome c reduction in the absence of CaM and of NO synthesis in its presence. Cytochrome c reduction by this chimera was inhibited by CaM. Other chimeras consisting of the nNOS heme domain, the CYPOR FMN binding subdomain, and the nNOS FAD binding subdomain with or without the tail region also catalyzed cytochrome c reduction, were not modulated by CaM, and could not transfer electrons into the heme domain. A chimera consisting of the heme domain of nNOS and the reductase domain of CYPOR reduced cytochrome c and ferricyanide at rates 2-fold higher than that of native CYPOR, suggesting that the presence of the heme domain affected electron transfer through the reductase domain. These data demonstrate that the FMN subdomain of CYPOR cannot effectively substitute for that of nNOS, whereas the FAD subdomains are interchangeable. The differences among these chimeras most likely result from alterations in the alignment of the flavins within each enzyme construct.     

Crystal structure of the FAD/NADPH-binding domain of rat neuronal nitric-oxide synthase. Comparisons with NADPH-cytochrome P450 oxidoreductase

Nitric-oxide synthase (NOS) is composed of a C-terminal, flavin-containing reductase domain and an N-terminal, heme-containing oxidase domain. The reductase domain, similar to NADPH-cytochrome P450 reductase, can be further divided into two different flavin-containing domains: (a) the N terminus, FMN-containing portion, and (b) the C terminus FAD- and NADPH-binding portion. The crystal structure of the FAD/NADPH-containing domain of rat neuronal nitric-oxide synthase, complexed with NADP(+), has been determined at 1.9 A resolution. The protein is fully capable of reducing ferricyanide, using NADPH as the electron donor. The overall polypeptide fold of the domain is very similar to that of the corresponding module of NADPH-cytochrome P450 oxidoreductase (CYPOR) and consists of three structural subdomains (from N to C termini): (a) the connecting domain, (b) the FAD-binding domain, and (c) the NADPH-binding domain. A comparison of the structure of the neuronal NOS FAD/NADPH domain and CYPOR reveals the strict conservation of the flavin-binding site, including the tightly bound water molecules, the mode of NADP(+) binding, and the aromatic residue that lies at the re-face of the flavin ring, strongly suggesting that the hydride transfer mechanisms in the two enzymes are very similar. In contrast, the putative FMN domain-binding surface of the NOS protein is less positively charged than that of its CYPOR counterpart, indicating a different nature of interactions between the two flavin domains and a different mode of regulation in electron transfer between the two flavins involving the autoinhibitory element and the C-terminal 33 residues, both of which are absent in CYPOR.     

SH3-dependent stimulation of Src-family kinase autophosphorylation without tail release from the SH2 domain in vivo

Src family protein-tyrosine kinase activity is suppressed by two intramolecular interactions. These involve binding of the SH2 domain to the phosphorylated C-terminal tail and association of the SH3 domain with a polyproline type II helix formed by the SH2-kinase linker. Here we show that SH3-dependent activation of the Src family member Hck by HIV-1 Nef binding or by SH2-kinase linker mutation does not affect tail tyrosine phosphorylation in fibroblasts. Surprisingly, replacement of the wild type Hck tail with a high-affinity SH2 domain-binding sequence did not affect Hck activation or downstream signaling by these SH3-dependent mechanisms, suggesting that activation through SH3 occurs without SH2-tail dissociation. These results identify SH3-linker interaction as an independent mode of Hck kinase regulation in vivo and suggest that different mechanisms of Src kinase activation may generate distinct output signals because of differences in SH2 or SH3 domain accessibility.     

Calmodulin-induced Conformational Control and Allostery Underlying Neuronal Nitric Oxide Synthase Activation

Nitric oxide synthase (NOS) is the primary generator of nitric oxide signals controlling diverse physiological processes such as neurotransmission and vasodilation. NOS activation is contingent on Ca^2 +/calmodulin binding at a linker between its oxygenase and reductase domains to induce large conformational changes that orchestrate inter-domain electron transfer. However, the structural dynamics underlying activation of full-length NOS remain ambiguous. Employing hydrogen–deuterium exchange mass spectrometry, we reveal mechanisms underlying neuronal NOS activation by calmodulin and regulation by phosphorylation. We demonstrate that calmodulin binding orders the junction between reductase and oxygenase domains, exposes the FMN subdomain, and elicits a more dynamic oxygenase active site. Furthermore, we demonstrate that phosphorylation partially mimics calmodulin activation to modulate neuronal NOS activity via long-range allostery. Calmodulin binding and phosphorylation ultimately promote a more dynamic holoenzyme while coordinating inter-domain communication and electron transfer.     

Chimeric forms of neuronal nitric oxide synthase identify different regions of the reductase domain that are essential for dimerization and activity.

Nitric oxide synthase (NOS) is the enzyme responsible for the conversion of L-arginine to L-citrulline and nitric oxide. Dimerization of the enzyme is an absolute requirement for catalytic activity. Each NOS monomer contains an N-terminal heme-binding domain and a C-terminal reductase domain. It is unclear how the reductase domain is involved in controlling dimerization and whether dimer formation alone controls enzyme activity. Our initial studies demonstrated that no dimerization or activity could be detected when the reductase domain of rat neuronal NOS (nNOS) was expressed either separately or in combination with the heme domain. To further evaluate the reductase domain, a set of expression plasmids was created by replacing the reductase domain of nNOS with other electron-transport proteins, thereby creating nNOS chimeric fusion proteins. The rat nNOS heme domain was linked with either cytochrome P450 reductase, adrenodoxin reductase, or the reductase domain from Bacillus megaterium cytochrome P450, BM-3. All the chimeric enzymes retained the ability to dimerize but were unable to metabolize L-arginine (<8% of wildtype activity levels), indicating that dimerization alone is insufficient to produce an active enzyme. Because the greatest regions of homology between electron-transport proteins are in the flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide phosphate (NADPH) binding regions, we produced truncation mutants within the nNOS reductase domain to investigate the role of these sequences in the ability of nNOS to dimerize and to metabolize L-arginine. The results demonstrated that the deletion of the final 56 amino acids or the NADPH-binding region had no effect on dimerization but produced an inactive enzyme. However, when the FAD-binding site (located between amino acids 920 and 1161) was deleted, both activity and dimerization were abolished. These results implicate sequences within the FAD-binding site as essential for nNOS dimerization but sequences within amino acids 1373 to 1429 as essential for activity.     

Reductase domain of Drosophila melanogaster nitric-oxide synthase: redox transformations, regulation, and similarity to mammalian homologues

The nitric oxide synthase of Drosophila melanogaster (dNOS) participates in essential developmental and behavioral aspects of the fruit fly, but little is known about dNOS catalysis and regulation. To address this, we expressed a construct comprising the dNOS reductase domain and its adjacent calmodulin (CaM) binding site (dNOSr) and characterized the protein regarding its catalytic, kinetic, and regulatory properties. The Ca2+ concentration required for CaM binding to dNOSr was between that of the mammalian endothelial and neuronal NOS enzymes. CaM binding caused the cytochrome c reductase activity of dNOSr to increase 4 times and achieve an activity comparable to that of mammalian neuronal NOS. This change was associated with decreased shielding of the FMN cofactor from solvent and an increase in the rate of NADPH-dependent flavin reduction. Flavin reduction in dNOSr was relatively slow following the initial 2-electron reduction, suggesting a slow inter-flavin electron transfer, and no charge-transfer complex was observed between bound NADP+ and reduced FAD during the process. We conclude that dNOSr catalysis and regulation is most similar to the mammalian neuronal NOS reductase domain, although differences exist in their flavin reduction behaviors. The apparent conservation between the fruit fly and mammalian enzymes is consistent with dNOS operating in various signal cascades that involve NO.     

Conformational basis for SH2-Tyr(P)527 binding in Src inactivation

Src protein-tyrosine kinase contains a myristoylation motif, a unique region, an Src homology (SH) 3 domain, an SH2 domain, a catalytic domain, and a C-terminal tail. The C-terminal tail contains a Tyr residue, Tyr527. Phosphorylation of Tyr527 triggers Src inactivation, caused by Tyr(P)527 binding to the SH2 domain. In this study, we demonstrated that a conformational contribution, not affinity, is the predominant force for the intramolecular SH2-Tyr(P)527 binding, and we characterized the structural basis for this conformational contribution. First, a phosphopeptide mimicking the C-terminal tail is an 80-fold weaker ligand than the optimal phosphopeptide, pYEEI, and similar to a phosphopeptide containing three Ala residues following Tyr(P) in binding to the Src SH2 domain. Second, the SH2-Tyr(P)527 binding is largely independent of the amino acid sequence surrounding Tyr(P)527, and only slightly decreased by an inactivating mutation in the SH2 domain. Furthermore, even the unphosphorylated C-terminal tail with the sequence of YEEI suppresses Src activity by binding to the SH2 domain. These experiments demonstrate that very weak affinity is sufficient for the SH2-Tyr(P)527 binding in Src inactivation. Third, the effective intramolecular SH2-Tyr(P)527 binding is attributed to a conformational contribution that requires residues Trp260 and Leu255. Although the SH3 domain is essential for Src inactivation by Tyr(P)527, it does not contribute to the SH2-Tyr(P)527 binding. These findings suggest a conformation-based Src inactivation model, which provides a unifying framework for understanding Src activation by a variety of mechanisms.     

Differences in a conformational equilibrium distinguish catalysis by the endothelial and neuronal nitric-oxide synthase flavoproteins

Nitric oxide (NO) is a physiological mediator synthesized by NO synthases (NOS). Despite their structural similarity, endothelial NOS (eNOS) has a 6-fold lower NO synthesis activity and 6-16-fold lower cytochrome c reductase activity than neuronal NOS (nNOS), implying significantly different electron transfer capacities. We utilized purified reductase domain constructs of either enzyme (bovine eNOSr and rat nNOSr) to investigate the following three mechanisms that may control their electron transfer: (i) the set point and control of a two-state conformational equilibrium of their FMN subdomains; (ii) the flavin midpoint reduction potentials; and (iii) the kinetics of NOSr-NADP+ interactions. Although eNOSr and nNOSr differed in their NADP(H) interaction and flavin thermodynamics, the differences were minor and unlikely to explain their distinct electron transfer activities. In contrast, calmodulin (CaM)-free eNOSr favored the FMN-shielded (electron-accepting) conformation over the FMN-deshielded (electron-donating) conformation to a much greater extent than did CaM-free nNOSr when the bound FMN cofactor was poised in each of its three possible oxidation states. NADPH binding only stabilized the FMN-shielded conformation of nNOSr, whereas CaM shifted both enzymes toward the FMN-deshielded conformation. Analysis of cytochrome c reduction rates measured within the first catalytic turnover revealed that the rate of conformational change to the FMN-deshielded state differed between eNOSr and nNOSr and was rate-limiting for either CaM-free enzyme. We conclude that the set point and regulation of the FMN conformational equilibrium differ markedly in eNOSr and nNOSr and can explain the lower electron transfer activity of eNOSr.     

Structure-function studies on nitric oxide synthases

Nitric oxide synthase (NOS) catalyzes the oxidation of one l-arginine guanidinium N atom to nitric oxide (NO). NOS consists of a heme domain linked to a flavin mononucleotide (FMN)/flavin adenine dinucleotide (FAD) reductase that shuttles electrons from nicotinamide adenine dinucleotide phosphate (NADPH) to the heme. This review summarizes various aspects of NOS structure and function derived from crystal structures coupled with a wealth of biochemical and biophysical data. This includes the binding of diatomic ligands, especially the product, NO, whose binding to the heme iron blocks enzyme activity. An unusual feature of NOS catalysis is the strict requirement for the essential cofactor, tetrahydrobiopterin (H4B). It now is generally agreed that H4B serves as an electron donor to the heme-oxy complex. The reason NOS may have recruited H4B as an electron transfer cofactor is to provide rapid coupled proton/electron transfer required for O2 activation. NOS is a highly regulated enzyme which is controlled by calmodulin (CaM) at the level of electron transfer within the FMN/FAD reductase and between the reductase and heme domains. Recent crystal structures provide a basis for developing models on the structural underpinnings of NOS regulation. In addition to the complex and fascinating functional and regulatory features of NOS, NOS is an important therapeutic target. Crystal structures have revealed the structural basis of isoform-selective inhibition by a group of dipeptide inhibitors which opens the way for structure-based inhibitor design.     

一氧化氮合酶FMN结合结构域的电子传递及调控机制研究现状

生物体内NO是在一氧化氮合酶（mitric oxide synthase,NOS）催化下生成的,NOS的结构包括C端还原酶域和N端加氧酶域。还原酶域中的FMN结合结构域既可接受来自NADPH-FAD结构域的电子,又可作为提供电子的供体,在调控催化过程中的电子传递方面发挥着重要作用。主要从FMN结合结构域的构象平衡及其对不同亚型NOS的动力学差异的贡献、FMN结合结构域自身的电荷性质以及NOS中其他结构域对FMN结构域的功能调控三个方面进行了论述,以期揭示NOS独特的电子传递催化机制。     

Reciprocal interference between the sequence-specific core and nonspecific C-terminal DNA binding domains of p53: implications for regulation.

The tumor suppressor p53 has two DNA binding domains: a central sequence-specific domain and a C-terminal sequence-independent domain. Here, we show that binding of large but not small DNAs by the C terminus of p53 negatively regulates sequence-specific DNA binding by the central domain. Four previously described mechanisms for activation of specific DNA binding operate by blocking negative regulation. Deletion of the C terminus of p53 activates specific DNA binding only in the presence of large DNA. Three activator molecules (a small nucleic acid, a monoclonal antibody against the p53 C terminus, and a C-terminal peptide of p53) stimulate sequence-specific DNA binding only in the presence of both large DNA and p53 with an intact C terminus. Our findings argue that interactions of the C terminus of p53 with genomic DNA in vivo would prevent p53 binding to specific promoters and that cellular mechanisms to block C-terminal DNA binding would be required.     

Azo reduction of methyl red by neuronal nitric oxide synthase: the important role of FMN in catalysis

Nitric oxide synthase (NOS) is composed of an oxygenase domain and a reductase domain. The reductase domain has NADPH, FAD, and FMN binding sites. Wild-type nNOS reduced the azo bond of methyl red with a turnover number of approximately 130 min(-1) in the presence of Ca(2+)/calmodulin (CaM) and NADPH under anaerobic conditions. Diphenyleneiodonium chloride (DPI), a flavin/NADPH binding inhibitor, completely inhibited azo reduction. The omission of Ca(2+)/CaM from the reaction system decreased the activity to 5%. The rate of the azo reduction with an FMN-deficient mutant was also 5% that of the wild type. NADPH oxidation rates for the wild-type and mutant enzymes were well coupled with azo reduction. Thus, we suggest that electrons delivered from the FMN of the nNOS enzyme reduce the azo bond of methyl red and that this reductase activity is controlled by Ca(2+)/CaM.