Endothelial nitric oxide synthase is regulated by ERK phosphorylation at Ser602.

eNOS (endothelial nitric oxide synthase) contains a MAPK (mitogen-activated protein kinase)-binding site associated with a major eNOS control element. Purified ERK (extracellular-signal-regulated kinase) phosphorylates eNOS with a stoichiometry of 2-3 phosphates per eNOS monomer. Phosphorylation decreases NO synthesis and cytochrome c reductase activity. Three sites of phosphorylation were detected by MS. All sites matched the SP and TP MAPK (mitogen-activated protein kinase) phosphorylation motif. Ser602 lies at the N-terminal edge of the 42-residue eNOS AI (autoinhibitory) element. The pentabasic MAPK-binding site lies at the opposite end of the AI, and other critical regulatory features are between them. Thr46 and Ser58 are located in a flexible region associated with the N terminus of the oxygenase domain. In contrast with PKC (protein kinase C), phosphorylation by ERK did not significantly interfere with CaM (calmodulin) binding as analysed by optical biosensing. Instead, ERK phosphorylation favours a state in which FMN and FAD are in close association and prevents conformational changes that expose reduced FMN to acceptors. The close associations between control sites in a few regions of the molecule suggest that control of signal generation is modulated by multiple inputs interacting directly on the surface of eNOS via overlapping binding domains and tightly grouped targets.

Calmodulin activates neuronal nitric oxide synthase by enabling transitions between conformational states.

We recently showed that inducible nitric oxide synthase conformational intermediates can be resolved via FMN fluorescence lifetimes. Here we show that neuronal NOS activation by calmodulin removes constraints favoring a closed 'input state', increasing occupation of other states and facilitating conformational transitions. The 90 ps FMN input state lifetime distinguishes it from ∼4 ns 'open' states in which FMN does not interact strongly with other groups, or 0.9 ns output states in which FMN interacts with ferriheme. Enablement of the conformational cycle is an important paradigm for control in nNOS and related enzymes, and may extend to other control modalities.

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 Å. These results indicate that FMN fluorescence is a valuable tool to study conformational states involved in the NOS reductase catalytic cycle.

Intraprotein electron transfer between the FMN and heme domains in endothelial nitric oxide synthase holoenzyme.

Intraprotein electron transfer (IET) from flavin mononucleotide (FMN) to heme is an essential step in nitric oxide (NO) synthesis by NO synthase (NOS). The IET kinetics in neuronal and inducible NOS (nNOS and iNOS) holoenzymes have been previously determined in our laboratories by laser flash photolysis [reviewed in: C.J. Feng, G. Tollin, Dalton Trans., (2009) 6692-6700]. Here we report the kinetics of the IET in a bovine endothelial NOS (eNOS) holoenzyme in the presence and absence of added calmodulin (CaM). The IET rate constant in the presence of CaM is estimated to be ~4.3s(-1). No IET was observed in the absence of CaM, indicating that CaM is the primary factor in controlling the FMN-heme IET in the eNOS enzyme. The IET rate constant value for the eNOS holoenzyme is approximately 10 times smaller than those obtained for the iNOS and CaM-bound nNOS holoenzymes. Possible mechanisms underlying the difference in IET kinetics among the NOS isoforms are discussed. Because the rate-limiting step in the IET process in these enzymes is the conformational change from input state to output state, a slower conformational change (than in the other isoforms) is most likely to cause the slower IET in eNOS.

Role of an isoform-specific substrate access channel residue in CO ligand accessibilities of neuronal and inducible nitric oxide synthase isoforms.

The rates of the bimolecular CO rebinding to the oxygenase domains of inducible and neuronal NOS proteins (iNOSoxy and nNOSoxy, respectively) after photolytic dissociation have been determined by laser flash photolysis. The following mutants at the isoform-specific sites (murine iNOSoxy N115L and rat nNOSoxy L337N, L337F) have been constructed to investigate role of the residues in the CO ligand accessibilities of the NOS isoforms. These residues are in the NOS distal substrate access channel. The effect of the (6R)-5,6,7,8-tetrahydrobiopterin (H(4)B) cofactor and l-arginine (Arg) substrate on the rates of CO rebinding have also been assessed. Addition of l-Arg to the iNOSoxy N115L mutant results in much faster CO rebinding rates, compared to the wild type. The results indicate that modifications to the iNOS channel in which the hydrophilic residue N115 is replaced by leucine (to resemble its nNOS cognate) open the channel somewhat, thereby improving access to the axial heme ligand binding position. On the other hand, introduction of a hydrophilic residue (L337N) or a bulky rigid aromatic residue (L337F) in the nNOS isoform does not significantly affect the kinetics profile, suggesting that the geometry of the substrate access pocket is not greatly altered. The bimolecular CO rebinding rate data indicate that the opening of the substrate access channel in the iNOS N115L mutant may be due to more widespread structural alterations induced by the mutation.

Electron transfer in a human inducible nitric oxide synthase oxygenase/FMN construct co-expressed with the N-terminal globular domain of calmodulin.

The FMN-heme intraprotein electron transfer (IET) kinetics in a human inducible NOS (iNOS) oxygenase/FMN (oxyFMN) construct co-expressed with NCaM, a truncated calmodulin (CaM) construct that includes only its N-terminal globular domain consisting of residues 1-75, were determined by laser flash photolysis. The IET rate constant is significantly decreased by nearly fourfold (compared to the iNOS oxyFMN co-expressed with full length CaM). This supports an important role of full length CaM in proper interdomain FMN/heme alignment in iNOS. The IET process was not observed with added excess EDTA, suggesting that Ca(2+) depletion results in the FMN domain moving away from the heme domain. The results indicate that a Ca(2+)-dependent reorganization of the truncated CaM construct could cause a major modification of the NCaM/iNOS association resulting in a loss of the IET.

Murine J774 macrophages recognize LPS/IFN-g, non-CpG DNA or two-CpG DNA-containing sequences as immunologically distinct.

Specific bacterial lipopolysaccharides (LPS), IFN-gamma, and unmethylated cytosine or guanosine-phosphorothioate containing DNAs (CpG) activate host immunity, influencing infectious responses. Macrophages detect, inactivate and destroy infectious particles, and synthetic CpG sequences invoke similar responses of the innate immune system. Previously, murine macrophage J774 cells treated with CpG induced the expression of nitric oxide synthase 2 (NOS2) and cyclo-oxygenase 2 (COX2) mRNA and protein. In this study murine J774 macrophages were exposed to vehicle, interferon gamma+lipopolysaccharide (IFN-g/LPS), non-CpG (SAK1), or two-CpG sequence-containing DNA (SAK2) for 0-18h and gene expression changes measured. A large number of immunostimulatory and inflammatory changes were observed. SAK2 was a stronger activator of TNFalpha- and chemokine expression-related changes than LPS/IFN-g. Up regulation included tumor necrosis factor receptor superfamily genes (TNFRSF's), IL-1 receptor signaling via stress-activated protein kinase (SAPK), NF-kappaB activation, hemopoietic maturation factors and sonic hedgehog/wingless integration site (SHH/Wnt) pathway genes. Genes of the TGF-beta pathway were down regulated. In contrast, LPS/IFN-g-treated cells showed increased levels for TGF-beta signaling genes, which may be linked to the observed up regulation of numerous collagens and down regulation of Wnt pathway genes. SAK1 produced distinct changes from LPS/IFN-g or SAK2. Therefore, J774 macrophages recognize LPS/IFN-g, non-CpG DNA or two-CpG DNA-containing sequences as immunologically distinct.

Space, time and nitric oxide--neuronal nitric oxide synthase generates signal pulses.

The temporal aspects of signaling are critical to the function of signals in communications, feedback regulation and control. The production and transduction of biological signals by enzymes comprises an area of central importance and rapid progress in the biomedical sciences. Treatment of signaling enzymes almost universally employs steady-state analyses that are suitable for mass catalysis but inappropriate for components in an information channel or a feedback/control system. In the present study, we show that, at 37 degrees C, neuronal nitric oxide synthase (EC 1.14.13.39) is progressively inhibited by the formation of an inhibited state during the first few turnovers (approximately 200 ms) after the initiation of catalysis, leading to pulse formation of nitric oxide. The general mechanism may be of wide importance in biological signaling.

Mutations in the FMN domain modulate MCD spectra of the heme site in the oxygenase domain of inducible nitric oxide synthase.

The nitric oxide synthase (NOS) output state for NO production is a complex of the flavin mononucleotide (FMN)-binding domain and the heme domain, and thereby it facilitates the interdomain electron transfer from the FMN to the catalytic heme site. Emerging evidence suggests that interdomain FMN-heme interactions are important in the formation of the output state because they guide the docking of the FMN domain to the heme domain. In this study, notable effects of mutations in the adjacent FMN domain on the heme structure in a human iNOS bidomain oxygenase/FMN construct have been observed by using low-temperature magnetic circular dichroism (MCD) spectroscopy. The comparative MCD study of wild-type and mutant proteins clearly indicates that a properly docked FMN domain contributes to the observed L-Arg perturbation of the heme MCD spectrum in the wild-type protein and that the conserved surface residues in the FMN domain (E546 and E603) play key roles in facilitating a productive alignment of the FMN and heme domains in iNOS.

Inhibition of nitric oxide synthase by cobalamins and cobinamides.

Cobalamins are important cofactors for methionine synthase and methylmalonyl-CoA mutase. Certain corrins also bind nitric oxide (NO), quenching its bioactivity. To determine if corrins would inhibit NO synthase (NOS), we measured their effects on -L-[(14)C]arginine-to-L-[(14)C]citrulline conversion by NOS1, NOS2, and NOS3. Hydroxocobalamin (OH-Cbl), cobinamide, and dicyanocobinamide (CN(2)-Cbi) potently inhibited all isoforms, whereas cyanocobalamin, methylcobalamin, and adenosylcobalamin had much less effect. OH-Cbl and CN(2)-Cbi prevented binding of the oxygen analog carbon monoxide (CO) to the reduced NOS1 and NOS2 heme active site. CN(2)-Cbi did not react directly with NO or CO. Spectral perturbation analysis showed that CN(2)-Cbi interacted directly with the purified NOS1 oxygenase domain. NOS inhibition by corrins was rapid and not reversed by dialysis with L-arginine or tetrahydrobiopterin. Molecular modeling indicated that corrins could access the unusually large heme- and substrate-binding pocket of NOS. Best fits were obtained in the "base-off" conformation of the lower axial dimethylbenzimidazole ligand. CN(2)-Cbi inhibited interferon-gamma-activated Raw264.7 mouse macrophage NO production. We show for the first time that certain corrins directly inhibit NOS, suggesting that these agents (or their derivatives) may have pharmacological utility. Endogenous cobalamins and cobinamides might play important roles in regulating NOS activity under normal and pathological conditions.

Intraprotein electron transfer in inducible nitric oxide synthase holoenzyme.

Intraprotein electron transfer (IET) from flavin mononucleotide (FMN) to heme is essential in NO synthesis by NO synthase (NOS). Our previous laser flash photolysis studies provided a direct determination of the kinetics of the FMN-heme IET in a truncated two-domain construct (oxyFMN) of murine inducible NOS (iNOS), in which only the oxygenase and FMN domains along with the calmodulin (CaM) binding site are present (Feng et al. J. Am. Chem. Soc. 128, 3808-3811, 2006). Here we report the kinetics of the IET in a human iNOS oxyFMN construct, a human iNOS holoenzyme, and a murine iNOS holoenzyme, using CO photolysis in comparative studies on partially reduced NOS and a NOS oxygenase construct that lacks the FMN domain. The IET rate constants for the human and murine iNOS holoenzymes are 34 +/- 5 and 35 +/- 3 s(-1), respectively, thereby providing a direct measurement of this IET between the catalytically significant redox couples of FMN and heme in the iNOS holoenzyme. These values are approximately an order of magnitude smaller than that in the corresponding iNOS oxyFMN construct, suggesting that in the holoenzyme the rate-limiting step in the IET is the conversion of the shielded electron-accepting (input) state to a new electron-donating (output) state. The fact that there is no rapid IET component in the kinetic traces obtained with the iNOS holoenzyme implies that the enzyme remains mainly in the input state. The IET rate constant value for the iNOS holoenzyme is similar to that obtained for a CaM-bound neuronal NOS holoenzyme, suggesting that CaM activation effectively removes the inhibitory effect of the unique autoregulatory insert in neuronal NOS.

Deletion of the autoregulatory insert modulates intraprotein electron transfer in rat neuronal nitric oxide synthase.

Comparative CO photolysis kinetics studies on wild-type and autoregulatory (AR) insert-deletion mutant of rat nNOS holoenzyme were conducted to directly investigate the role of the unique AR insert in the catalytically significant FMN-heme intraprotein electron transfer (IET). Although the amplitude of the IET kinetic traces was decreased two- to three-fold, the AR deletion did not change the rate constant for the calmodulin-controlled IET. This suggests that the rate-limiting conversion of the electron-accepting state to a new electron-donating (output) state does not involve interactions with the AR insert, but that AR may stabilize the output state once it is formed.

CLL cell apoptosis induced by nitric oxide synthase inhibitors: correlation with lipid solubility and NOS1 dissociation constant.

Nitric oxide synthase (NOS) inhibitors induce chronic lymphocytic leukemia (CLL) cell apoptosis and have potential as CLL therapeutics. We determined the half-maximal concentration (ED(50)) of 22 NOS inhibitors that induced CLL cell death in vitro. There was a direct correlation of the NOS1 (but not NOS2) dissociation constant (K(d)) and the hydrophobicity partitioning coefficient of each NOS inhibitor and its ED(50). NOS inhibitors that bound tightly to CLL cell NOS1 and were hydrophobic potently induced CLL cell death. CLL cell RNA and protein analyses confirmed CLL cell NOS1 expression. Our studies permit the rational selection of NOS inhibitors for testing as CLL therapeutics.

Direct measurement by laser flash photolysis of intraprotein electron transfer in a rat neuronal nitric oxide synthase.

Intraprotein interdomain electron transfer (IET) from flavin mononucleotide (FMN) to heme is essential in nitric oxide (NO) synthesis by NO synthase (NOS). Our previous laser flash photolysis studies have provided a direct determination of the kinetics of IET between the FMN and heme domains in truncated oxyFMN constructs of rat neuronal NOS (nNOS) and murine inducible NOS (iNOS), in which only the oxygenase and FMN domains along with the calmodulin (CaM) binding site are present [Feng, C. J.; Tollin, G.; Holliday, M. A.; Thomas, C.; Salerno, J. C.; Enemark, J. H.; Ghosh, D. K. Biochemistry 2006, 45, 6354-6362. Feng, C. J.; Thomas, C.; Holliday, M. A.; Tollin, G.; Salerno, J. C.; Ghosh, D. K.; Enemark, J. H. J. Am. Chem. Soc. 2006, 128, 3808-3811]. Here, we report the kinetics of IET between the FMN and heme domains in a rat nNOS holoenzyme in the presence and absence of added CaM using laser flash photolysis of CO dissociation in comparative studies on partially reduced NOS and a single domain NOS oxygenase construct. The IET rate constant in the presence of CaM is 36 s-1, whereas no IET was observed in the absence of CaM. The kinetics reported here are about an order of magnitude slower than the kinetics in a rat nNOS oxyFMN construct with added CaM (262 s-1). We attribute the slower IET between FMN and heme in the holoenzyme to the additional step of dissociation of the FMN domain from the reductase complex before reassociation with the oxygenase domain to form the electron-transfer competent output state complex. This work provides the first direct measurement of CaM-controlled electron transfer between catalytically significant redox couples of FMN and heme in a nNOS holoenzyme.

Intraprotein electron transfer in a two-domain construct of neuronal nitric oxide synthase: the output state in nitric oxide formation.

Intersubunit intraprotein electron transfer (IET) from flavin mononucleotide (FMN) to heme is essential in nitric oxide (NO) synthesis by NO synthase (NOS). Previous crystal structures and functional studies primarily concerned an enzyme conformation, which serves as the input state for reduction of FMN by electrons from NADPH and flavin adenine dinucleotide (FAD) in the reductase domain. To favor the formation of the output state for the subsequent IET from FMN to heme in the oxygenase domain, a novel truncated two-domain oxyFMN construct of rat neuronal NOS (nNOS), in which only the FMN and heme domains were present, was designed and expressed. The kinetics of IET between the FMN and heme domains in the nNOS oxyFMN construct in the presence and absence of added calmodulin (CaM) were directly determined using laser flash photolysis of CO dissociation in comparative studies on partially reduced oxyFMN and single-domain heme oxygenase constructs. The IET rate constant in the presence of CaM (262 s(-)(1)) was increased approximately 10-fold compared to that in the absence of CaM (22 s(-)(1)). The effect of CaM on interdomain interactions was further evidenced by electron paramagnetic resonance (EPR) spectra. This work provides the first direct evidence of the CaM control of electron transfer (ET) between FMN and heme domains through facilitation of the FMN/heme interactions in the output state. Therefore, CaM controls IET between heme and FMN domains by a conformational gated mechanism. This is essential in coupling ET in the reductase domain in NOS with NO synthesis in the oxygenase domain.

Binding and activation of nitric oxide synthase isozymes by calmodulin EF hand pairs.

Calmodulin (CaM) is a cytosolic Ca(2+) signal-transducing protein that binds and activates many different cellular enzymes with physiological relevance, including the nitric oxide synthase (NOS) isozymes. CaM consists of two globular domains joined by a central linker; each domain contains an EF hand pair. Four different mutant CaM proteins were used to investigate the role of the two CaM EF hand pairs in the binding and activation of the mammalian inducible NOS (iNOS) and the constitutive NOS (cNOS) enzymes, endothelial NOS (eNOS) and neuronal NOS (nNOS). The role of the CaM EF hand pairs in different aspects of NOS enzymatic function was monitored using three assays that monitor electron transfer within a NOS homodimer. Gel filtration studies were used to determine the effect of Ca(2+) on the dimerization of iNOS when coexpressed with CaM and the mutant CaM proteins. Gel mobility shift assays were performed to determine binding stoichiometries of CaM proteins to synthetic NOS CaM-binding domain peptides. Our results show that the N-terminal EF hand pair of CaM contains important binding and activating elements for iNOS, whereas the N-terminal EF hand pair in conjunction with the central linker region is required for cNOS enzyme binding and activation. The iNOS enzyme must be coexpressed with wild-type CaM in vitro because of its propensity to aggregate when residues of the highly hydrophobic CaM-binding domain are exposed to an aqueous environment. A possible role for iNOS aggregation in vivo is also discussed.

Direct measurement by laser flash photolysis of intramolecular electron transfer in a two-domain construct of murine inducible nitric oxide synthase.

Intersubunit intramolecular electron transfer (IET) from FMN to heme is essential in the delivery of electrons required for O2 activation in the heme domain and the subsequent nitric oxide (NO) synthesis by NO synthase (NOS). Previous crystal structures and functional studies primarily concerned an enzyme conformation that serves as the input state for reduction of FMN by electrons from NADPH and FAD in the reductase domain. To favor formation of the output state for the subsequent IET from FMN to heme in the oxygenase domain, a novel truncated two-domain oxyFMN construct murine inducible nitric oxide synthase (iNOS), in which only the FMN and heme domains were present, was designed and expressed. The kinetics of the IET between the FMN and heme domains in this construct was directly determined using laser flash photolysis of CO dissociation in comparative studies on partially reduced oxyFMN and single domain heme oxygenase constructs.

Nitric-oxide synthase output state. Design and properties of nitric-oxide synthase oxygenase/FMN domain constructs.

Mammalian nitric-oxide synthases are large modular enzymes that evolved from independently expressed ancestors. Calmodulin-controlled isoforms are signal generators; calmodulin activates electron transfer from NADPH through three reductase domains to an oxygenase domain. Structures of the reductase unit and its homologs show FMN and FAD in contact but too isolated from the protein surface to permit exit of reducing equivalents. To study states in which FMN/heme electron transfer is feasible, we designed and produced constructs including only oxygenase and FMN binding domains, eliminating strong internal reductase complex interactions. Constructs for all mammalian isoforms were expressed and purified as dimers. All synthesize NO with peroxide as the electron donor at rates comparable with corresponding oxygenase constructs. All bind cofactors nearly stoichiometrically and have native catalytic sites by spectroscopic criteria. Modest differences in electrochemistry versus independently expressed heme and FMN binding domains suggest interdomain interactions. These interactions can be convincingly demonstrated via calmodulin-induced shifts in high spin ferriheme EPR spectra and through mutual broadening of heme and FMNH. radical signals in inducible nitric-oxide synthase constructs. Blue neutral FMN semiquinone can be readily observed; potentials of one electron couple (in inducible nitric-oxide synthase oxygenase FMN, FMN oxidized/semiquinone couple = +70 mV, FMN semiquinone/hydroquinone couple = -180 mV, and heme = -180 mV) indicate that FMN is capable of serving as a one electron heme reductant. The construct will serve as the basis for future studies of the output state for NADPH derived reducing equivalents.

Structural analysis of isoform-specific inhibitors targeting the tetrahydrobiopterin binding site of human nitric oxide synthases.

Nitric oxide synthesized from l-arginine by nitric oxide synthase isoforms (NOS-I-III) is physiologically important but also can be deleterious when overproduced. Selective NOS inhibitors are of clinical interest, given their differing pathophysiological roles. Here we describe our approach to target the unique NOS (6R,1'R,2'S)-5,6,7,8-tetrahydrobiopterin (H(4)Bip) binding site. By a combination of ligand- and structure-based design, the structure-activity relationship (SAR) for a focused set of 41 pteridine analogues on four scaffolds was developed, revealing selective NOS-I inhibitors. The X-ray crystal structure of rat NOS-I dimeric-oxygenase domain with H(4)Bip and l-arginine was determined and used for human isoform homology modeling. All available NOS structural information was subjected to comparative analysis of favorable protein-ligand interactions using the GRID/concensus principal component analysis (CPCA) approach to identify the isoform-specific interaction site. Our interpretation, based on protein structures, is in good agreement with the ligand SAR and thus permits the rational design of next-generation inhibitors targeting the H(4)Bip binding site with enhanced isoform selectivity for therapeutics in pathology with NO overproduction.

Differential activation of nitric-oxide synthase isozymes by calmodulin-troponin C chimeras.

The interactions of neuronal nitric-oxide synthase (nNOS) with calmodulin (CaM) and mutant forms of CaM, including CaM-troponin C chimeras, have been previously reported, but there has been no comparable investigation of CaM interactions with the other constitutively expressed NOS (cNOS), endothelial NOS (eNOS), or the inducible isoform (iNOS). The present study was designed to evaluate the role of the four CaM EF hands in the activation of eNOS and iNOS. To assess the role of CaM regions on aspects of enzymatic function, three distinct activities associated with NOS were measured: NADPH oxidation, cytochrome c reduction, and nitric oxide (*NO) generation as assessed by the oxyhemoglobin capture assay. CaM activates the cNOS enzymes by a mechanism other than stimulating electron transfer into the oxygenase domain. Interactions with the reductase moiety are dominant in cNOS activation, and EF hand 1 is critical for activation of both nNOS and eNOS. Although the activation patterns for nNOS and eNOS are clearly related, effects of the chimeras on all the reactions are not equivalent. We propose that cytochrome c reduction is a measure of the release of the FMN domain from the reductase complex. In contrast, cytochrome c reduction by iNOS is readily activated by each of the chimeras examined here and may be constitutive. Each of the chimeras were co-expressed with the human iNOS enzyme in Escherichia coli and subsequently purified. Domains 2 and 3 of CaM contain important elements required for the Ca2+/CaM independence of *NO production by the iNOS enzyme. The disparity between cytochrome c reduction and *NO production at low calcium can be attributed to poor association of heme and FMN domains when the bound CaM constructs are depleted of Ca2+. In general cNOSs are much more difficult to activate than iNOS, which can be attributed to their extra sequence elements, which are adjacent to the CaM-binding site and associated with CaM control.