Identification of imidazole as l-arginine-competitive inhibitor of porcine brain nitric oxide synthase

Imidazole acts as a heme-site inhibitor of nitric oxide synthase (NOS). We used this compound to investigate whether the substrate l-arginine binds directly to the heme or to a separate domain of brain NOS. Enzyme kinetic experiments showed that imidazole enhanced the apparent K_m for l-arginine without affecting maximal enzyme activity, and binding studies revealed that the inhibitor displaced the radioligand N^G-nitro-l-[³H]arginine in a concentration-dependent fashion. These results demonstrate that imidazole exerts its effects on NOS in an l-arginine-competitive manner and that the substrate site of the enzyme may be identical with the prosthetic heme group.     

How does a valine residue that modulates heme-NO binding kinetics in inducible NO synthase regulate enzyme catalysis?

Nitric oxide (NO) release from nitric oxide synthases (NOSs) depends on the dissociation of a ferric heme-NO product complex (Fe^IIINO) that forms immediately after NO is made in the heme pocket. The NOS-like enzyme of Bacillus subtilis (bsNOS) has 10-20 fold slower Fe^IIINO dissociation rate (k_d) and NO association rate (k_on) compared to mammalian NOS counterparts. We previously showed that an Ile for Val substitution at the opening of the heme pocket in bsNOS contributes to these differences. The complementary mutation in mouse inducible NOS oxygenase domain (Val346Ile) decreased the NO k_on and k_d by 8 and 3-fold, respectively, compared to wild-type iNOSoxy, and also slowed the reductive processing of the heme-O₂ catalytic intermediate. To investigate how these changes affect steady-state catalytic behaviors, we generated and characterized the V346I mutant of full-length inducible NOS (iNOS). The mutant exhibited a 4-5 fold lower NO synthesis activity, an apparent uncoupled NADPH consumption, and formation of a heme-NO complex during catalysis that was no longer sensitive to solution NO scavenging. We found that these altered catalytic behaviors were not due to changes in the heme reduction rate or in the stability of the enzyme heme-O₂ intermediate, but instead were due to the slower NO k_on and k_d and a slower oxidation rate of the enzyme ferrous heme-NO complex. Computer simulations that utilized the measured kinetic values confirmed this interpretation, and revealed that the V346I iNOS has an enhanced NADPH-dependent NO dioxygenase activity that converts almost 1 NO to nitrate for every NO that the enzyme releases into solution. Together, our results highlight the importance of heme pocket geometry in tuning the NO release versus NO dioxygenase activities of iNOS.     

Role of Arginine Guanidinium Moiety in Nitric-oxide Synthase Mechanism of Oxygen Activation

Nitric-oxide synthases (NOS) are highly regulated heme-thiolate enzymes that catalyze two oxidation reactions that sequentially convert the substrate l-Arg first to N^ω-hydroxyl-l-arginine and then to l-citrulline and nitric oxide. Despite numerous investigations, the detailed molecular mechanism of NOS remains elusive and debatable. Much of the dispute in the various proposed mechanisms resides in the uncertainty concerning the number and sources of proton transfers. Although specific protonation events are key features in determining the specificity and efficiency of the two catalytic steps, little is known about the role and properties of protons from the substrate, cofactors, and H-bond network in the vicinity of the heme active site. In this study, we have investigated the role of the acidic proton from the l-Arg guanidinium moiety on the stability and reactivity of the ferrous heme-oxy complex intermediate by exploiting a series of l-Arg analogues exhibiting a wide range of guanidinium pK_a values. Using electrochemical and vibrational spectroscopic techniques, we have analyzed the effects of the analogues on the heme, including characteristics of its proximal ligand, heme conformation, redox potential, and electrostatic properties of its distal environment. Our results indicate that the substrate guanidinium pK_a value significantly affects the H-bond network near the heme distal pocket. Our results lead us to propose a new structural model where the properties of the guanidinium moiety finely control the proton transfer events in NOS and tune its oxidative chemistry. This model may account for the discrepancies found in previously proposed mechanisms of NOS oxidation processes.     

Comparison of wild type neuronal nitric oxide synthase and its Tyr588Phe mutant towards various l-arginine analogues

Crystal structures of nitric oxide synthases (NOS) isoforms have shown the presence of a strongly conserved heme active-site residue, Tyr588 (numbering for rat neuronal NOS, nNOS). Preliminary biochemical studies have highlighted its importance in the binding and oxidation to NO of natural substrates L-Arg and N^ω-hydroxy-l-arginine (NOHA) and suggested its involvement in mechanism. We have used UV-visible and EPR spectroscopy to investigate the effects of the Tyr588 to Phe mutation on the heme-distal environment, on the binding of a large series of guanidines and N-hydroxyguanidines that differ from L-Arg and NOHA by the nature of their alkyl- or aryl-side chain, and on the abilities of wild type (WT) and mutant to oxidize these analogues with formation of NO. Our EPR experiments show that the heme environment of the Tyr588Phe mutant differs from that of WT nNOS. However, the addition of L-Arg to this mutant results in EPR spectra similar to that of WT nNOS. Tyr588Phe mutant binds L-Arg and NOHA with much weaker affinities than WT nNOS but both proteins bind non α-amino acid guanidines and N-hydroxyguanidines with close affinities. WT nNOS and mutant do not form NO from the tested guanidines but oxidize several N-hydroxyguanidines with formation of NO in almost identical rates. Our results show that the Tyr588Phe mutation induces structural modifications of the H-bonds network in the heme-distal site that alter the reactivity of the heme. They support recent spectroscopic and mechanistic studies that involve two distinct heme-based active species in the two steps of NOS mechanism.     

Insights into the structural determinants for selective inhibition of nitric oxide synthase isoforms

Selective inhibition of the nitric oxide synthase isoforms (NOS) is a promising approach for the treatment of various disorders. However, given the high active site conservation among all NOS isoforms, the design of selective inhibitors is a challenging task. Analysis of the X-ray crystal structures of the NOS isoforms complexed with known inhibitors most often gives no clues about the structural determinants behind the selective inhibition since the inhibitors share the same binding conformation. Aimed at a better understanding of the structural factors responsible for selective inhibition of NOS isoforms we have performed MD simulations for iNOS, nNOS and eNOS complexed with N^ω-NO₂-L-Arg (1), and with the aminopyridine derivatives 2 and 3. The slightly better selectivity of 1 for nNOS may be assigned to the presence of extra charge–charge interactions due to its “extended” conformation. While the high affinity of 2 for iNOS can be explained by the formation of an iNOS-specific subpocket upon binding, the lack of affinity for eNOS is associated to a conformational change in Glu363. The strong van der Waals and electrostatic interactions between 3 and the active site of nNOS are most likely responsible for its higher affinity for this isoform. Owing to the elongated and narrow binding pocket of iNOS, the correct positioning of 3 over the heme group is difficult, which may account for its lower affinity toward this isoform. Brought together, our results might help to rationalize the design of selective NOS inhibitors.

The Involvement of Mitochondrial Amidoxime Reducing Components 1 and 2 and Mitochondrial Cytochrome b5 in N-Reductive Metabolism in Human Cells

The mitochondrial amidoxime reducing component mARC is a recently discovered molybdenum enzyme in mammals. mARC is not active as a standalone protein, but together with the electron transport proteins NADH-cytochrome b₅ reductase (CYB5R) and cytochrome b₅ (CYB5), it catalyzes the reduction of N-hydroxylated compounds such as amidoximes. The mARC-containing enzyme system is therefore considered to be responsible for the activation of amidoxime prodrugs. All hitherto analyzed mammalian genomes code for two mARC genes (also referred to as MOSC1 and MOSC2), which share high sequence similarities. By RNAi experiments in two different human cell lines, we demonstrate for the first time that both mARC proteins are capable of reducing N-hydroxylated substrates in cell metabolism. The extent of involvement is highly dependent on the expression level of the particular mARC protein. Furthermore, the mitochondrial isoform of CYB5 (CYB5B) is clearly identified as an essential component of the mARC-containing N-reductase system in human cells. The participation of the microsomal isoform (CYB5A) in N-reduction could be excluded by siRNA-mediated down-regulation in HEK-293 cells and knock-out in mice. Using heme-free apo-CYB5, the contribution of mitochondrial CYB5 to N-reductive catalysis was proven to strictly depend on heme. Finally, we created recombinant CYB5B variants corresponding to four nonsynonymous single nucleotide polymorphisms (SNPs). Investigated mutations of the heme protein seemed to have no significant impact on N-reductive activity of the reconstituted enzyme system.     

Nanosecond photoreduction of inducible nitric oxide synthase by a Ru-diimine electron tunneling wire bound distant from the active site

A Ru-diimine wire, [(4,4′,5,5′-tetramethylbipyridine)₂Ru(F₉bp)]²⁺ (tmRu-F₉bp, where F₉bp is 4-methyl-4′-methylperfluorobiphenylbipyridine), binds tightly to the oxidase domain of inducible nitric oxide synthase (iNOSoxy). The binding of tmRu-F₉bp is independent of tetrahydrobiopterin, arginine, and imidazole, indicating that the wire resides on the surface of the enzyme, distant from the active-site heme. Photoreduction of an imidazole-bound active-site heme iron in the enzyme-wire conjugate (k_ET = 2(1) × 10⁷ s⁻¹) is fully seven orders of magnitude faster than the in vivo process.     

Atrial natriuretic peptide inhibits nitric oxide synthesis in mouse macrophages

The atrial natriuretic peptide (ANP) affects cardiovascular physiology, and, as has been suggested more recently, exerts immunomodulatory activities. In this context, we examined the effect of ANP on nitric oxide (NO) synthesis in murine bone marrow derived macrophages as well as in peritoneal macrophages. Cultured macrophages were stimulated with lipopolysaccharides (LPS 0.1–10 μg/ml) and NO synthesis was monitored by measuring increased concentrations of NO₂ in the medium. In initial experiments employment of N^G-monomethyl-L-arginine (L-NMMA) and dexamethasone, two specific inhibitors of nitric oxide synthase (NOS), confirmed the presence of inducible NOS activity in the cells. Exposure of cells to rat ANP99–126 in the range of 10⁻⁸ to 10⁻⁶ M significantly decreased LPS induced NO synthesis over 24 hours of incubation. Thus, ANP may alter macrophage function by affecting their nitric oxide synthesizing pathway.     

Expression,purification and characterization of an active C491G variant of ferredoxin sulfite reductase from Synechococcus elongatus PCC 7942

Recently developed molecular wire technology takes advantage of [4Fe-4S] clusters that are ligated by at least one surface exposed Cys residue. Mutagenesis of this Cys residue to a Gly opens an exchangeable coordination site to a corner iron atom that can be chemically rescued by an external thiolate ligand. This ligand can be subsequently displaced by mass action using a dithiol molecular wire to tether two redox active proteins. We intend to apply this technique to tethering Photosystem I to ferredoxin sulfite reductase (FdSiR), an enzyme that catalyzes the six-electron reduction of sulfite to hydrogen sulfite and nitrite to ammonia. The enzyme contains a [4Fe-4S]^2+/1+ cluster and a siroheme active site. FdSiR_WT and an FdSiR_C491G variant were cloned from Synechococcus elongatus PCC 7942 and expressed along with the cysG gene from Salmonella typhimurium using the pCDFDuet plasmid. UV/Vis absorbance spectra of both FdSiR_WT and the FdSiR_C491G variant displayed characteristic peaks at 278, 392 (Soret), 585 (α) and 714?nm (charge transfer band), and 278, 394 (Soret), 587 (α) and 714?nm (charge transfer band) respectively. Both enzymes in their as-isolated forms displayed an EPR spectrum characteristic of an S?=?5/2 high spin heme. When reduced, both enzymes exhibited the signal of a low spin S?=?1/2 [4Fe-4S]¹⁺ cluster. The FdSiR_WT and FdSiR_C491G variant both showed activity using reduced methyl viologen and Synechococcus elongatus PCC 7942 ferredoxin 1 (Fd1) as electron donors. Based on these results, the FdSIR_C491G variant should be a suitable candidate for wiring to Photosystem I.     

Multidomain Human Peroxidasin 1 Is a Highly Glycosylated and Stable Homotrimeric High Spin Ferric Peroxidase

Human peroxidasin 1 (hsPxd01) is a multidomain heme peroxidase that uses bromide as a cofactor for the formation of sulfilimine cross-links. The latter confers critical structural reinforcement to collagen IV scaffolds. Here, hsPxd01 and various truncated variants lacking nonenzymatic domains were recombinantly expressed in HEK cell lines. The N-glycosylation site occupancy and disulfide pattern, the oligomeric structure, and unfolding pathway are reported. The homotrimeric iron protein contains a covalently bound ferric high spin heme per subunit with a standard reduction potential of the Fe(III)/Fe(II) couple of −233 ± 5 mV at pH 7.0. Despite sequence homology at the active site and biophysical properties similar to human peroxidases, the catalytic efficiency of bromide oxidation (k_cat/K_M^app) of full-length hsPxd01 is rather low but increased upon truncation. This is discussed with respect to its structure and proposed biosynthetic function in collagen IV cross-linking.     

Implications for isoform-selective inhibitor design derived from the binding mode of bulky isothioureas to the heme domain of endothelial nitric-oxide synthase

Nitric oxide produced by nitric-oxide synthase (NOS) is not only involved in a wide range of physiological functions but also in a variety of pathological conditions. Isoform-selective NOS inhibitors are highly desirable to regulate the NO production of one isoform beneficial to normal physiological functions from the uncontrolled NO production of another isoform that accompanies certain pathological states. Crystal structures of the heme domain of the three NOS isoforms have revealed a very high degree of similarity in the immediate vicinity of the heme active site illustrating the challenge of isoform-selective inhibitor design. Isothioureas are potent NOS inhibitors, and the structures of the endothelial NOS heme domain complexed with isothioureas bearing small S-alkyl substituents have been determined (Li, H., Raman, C.S., Martásek, P., Král, V., Masters, B.S.S., and Poulos, T.L. (2000) J. Inorg. Biochem. 81, 133--139). In the present communication, the binding mode of larger bisisothioureas complexed to the endothelial NOS heme domain has been determined. These structures afford a structural rationale for the known inhibitory activities. In addition, these structures provide clues on how to exploit the longer inhibitor substituents that extend out of the active site pocket for isoform-selective inhibitor design.     

The Characteristics and Regulatory Mechanisms of Superoxide Generation from eNOS Reductase Domain

In addition to superoxide (O₂ ^.-) generation from nitric oxide synthase (NOS) oxygenase domain, a new O₂ ^.- generation site has been identified in the reductase domain of inducible NOS (iNOS) and neuronal NOS (nNOS). Cysteine S-glutathionylation in eNOS reductase domain also induces O₂ ^.- generation from eNOS reductase domain. However, the characteristics and regulatory mechanism of the O₂ ^.- generation from NOS reductase domain remain unclear. We cloned and purified the wild type bovine eNOS (WT eNOS), a mutant of Serine 1179 replaced with aspartic acid eNOS (S1179D eNOS), which mimics the negative charge caused by phosphorylationand truncated eNOS reductase domain (eNOS RD). Both WT eNOS and S1179D eNOS generated significant amount of O₂ ^.- in the absence of BH4 and L-arginine. The capacity of O₂ ^.- generation from S1179D eNOS was significantly higher than that of WT eNOS (1.74:1). O₂ ^.- generation from both WT eNOS and S1179D eNOS were not completely inhibited by 100nM tetrahydrobiopterin(BH4). This BH4 un-inhibited O₂ ^.- generation from eNOS was blocked by 10mM flavoprotein inhibitor, diphenyleneiodonium (DPI). Purified eNOS reductase domain protein confirmed that this BH4 un-inhibited O₂ ^.- generation originates at the FMN or FAD/NADPH binding site of eNOS reductase domain. DEPMPO-OOH adduct EPR signals and NADPH consumptions analyses showed that O₂ ^.- generation from eNOS reductase domain was regulated by Serine 1179 phosphorylation and DPI, but not by L-arginine, BH4 or calmodulin (CaM). In addition to the heme center of eNOS oxygenase domain, we confirmed another O₂ ^.- generation site in the eNOS reductase domain and characterized its regulatory properties.     

Retinoic acid-induced differentiation in a human neuroblastoma cell line is associated with an increase in nitric oxide synthesis

The human neuroblastoma cell line SK-N-BE, after incubation with 10 μM retinoic acid (RA) or 20 nM phorbol 12-myristate 13-acetate (PMA), underwent biochemical and morphological signs of differentiation within 10–14 days. In parallel, SK-N-BE cells produced significantly higher amounts of nitric oxide (NO) in comparison with controls, as assessed by the measurement of nitrite and nitrate in the culture supernatant and of NO synthase (NOS) activity in the cell lysates (measured as ability to convert [³H]arginine into [³H]citrulline and as NADPH diaphorase activity). Nitrite/nitrate production was abolished by adding the NO scavenger hemoglobin in the culture medium and was inhibited by aminoguanidine (AG, a selective inhibitor of the inducible NOS isoform) but not by the less selective inhibitor N^G-nitro-L -arginine methylester (NAME). Western blotting experiments with monoclonal antibodies against the ncNOS and iNOS isoforms suggest that RA-elicited NOS activation is not attributable to an increased expression of the protein. NAME and AG were not able to revert inhibition of proliferation induced by RA, and the NO donor sodium nitroprusside did not mimic the effect of RA and PMA. These data indicate that increased NO synthesis does not mediate RA- or PMA-induced differentiation but may be an additional marker of differentiation into sympathetic-like neuronal cells. J. Cell. Physiol. 174:99–106, 1998. © 1998 Wiley-Liss, Inc.     

Substrate Control of Internal Electron Transfer in Bacterial Nitric-oxide Reductase

Nitric -oxide reductase (NOR) from Paracoccus denitrificans catalyzes the reduction of nitric oxide (NO) to nitrous oxide (N₂O) (2NO + 2H⁺ + 2e⁻ →N₂O + H₂O) by a poorly understood mechanism. NOR contains two low spin hemes c and b, one high spin heme b₃, and a non-heme iron Fe_B. Here, we have studied the reaction between fully reduced NOR and NO using the “flow-flash” technique. Fully (four-electron) reduced NOR is capable of two turnovers with NO. Initial binding of NO to reduced heme b₃ occurs with a time constant of ∼1 μs at 1.5 mm NO, in agreement with earlier studies. This reaction is [NO]-dependent, ruling out an obligatory binding of NO to Fe_B before ligation to heme b₃. Oxidation of hemes b and c occurs in a biphasic reaction with rate constants of 50 s⁻¹ and 3 s⁻¹ at 1.5 mm NO and pH 7.5. Interestingly, this oxidation is accelerated as [NO] is lowered; the rate constants are 120 s⁻¹ and 12 s⁻¹ at 75 μm NO. Protons are taken up from solution concomitantly with oxidation of the low spin hemes, leading to an acceleration at low pH. This effect is, however, counteracted by a larger degree of substrate inhibition at low pH. Our data thus show that substrate inhibition in NOR, previously observed during multiple turnovers, already occurs during a single oxidative cycle. Thus, NO must bind to its inhibitory site before electrons redistribute to the active site. The further implications of our data for the mechanism of NO reduction by NOR are discussed.     

Determination of sialidase activities in HeLa cells using gangliosides specifically labeled in N-acetylneuraminic acid.

Radioactive gangliosides, N-[¹⁴C]-acetylneuraminylgalactosylglucosylceramide ([¹⁴C]G_M3) and N- [¹⁴C]-acetylneuraminylgalactosyl-N-acetylgalactosaminyl- [N-acetylneuraminyl]-galactosylglucosylceramide ([¹⁴C]G_D1a), were synthesized from CMP-[¹⁴C]sialic acid and the appropriate precursor glycolipid using specific sialyltransferase activities. These compounds were isolated and used as substrates to assay sialidase activity in HeLa cells. Although sodium butyrate added to the culture medium increased G_M3 biosynthesis in HeLa cells, sialidase activity, as well as that of other glycohydrolases, was the same in control and butyrate-treated HeLa cells. The same sialidase activity appeared to hydrolyze both [¹⁴C]G_M3 and [¹⁴C]G_D1a, but not fetuin; the enzyme had a pH optimum of 5.0 and a K_m of 75 μm for the ganglioside substrates. Although the cells contained a high sialidase activity (4–7 nmol/mg of protein/h) and could bind exogenously added [¹⁴C]G_M3, no “ecto”-sialidase activity would be detected in intact cells under conditions where a close to physiological pH is maintained. The results indicate that ganglioside sialidase is not involved directly in the morphological and biochemical differentiation induced in HeLa cells by exposure to sodium butyrate.     

N-Hydroxyguanidines oxidation by a N3S copper-complex mimicking the reactivity of Dopamine β-Hydroxylase

N-Aryl-N′-hydroxyguanidines are compounds that display interesting pharmacological properties but their chemical reactivity remains poorly investigated. Some of these compounds are substrates for the heme-containing enzymes nitric-oxide synthases (NOS) and act as reducing co-substrates for the copper-containing enzyme Dopamine β-Hydroxylase (DBH) [P. Slama, J.L. Boucher, M. Réglier, Biochem. Biophys. Res. Commun. 316 (2004) 1081-1087]. DBH catalyses the hydroxylation of the important neurotransmitter dopamine into norepinephrine in the presence of both molecular oxygen and a reducing co-substrate. Although many molecules have been used as co-substrates for DBH, their interaction at the active site of DBH and their role in mechanism are not clearly characterized. In the present paper, we have used a water-soluble copper-N₃S complex that mimics the Cu_B site of DBH, and aromatic N-hydroxyguanidines as reducers to address this question. N-Aryl-N′-hydroxyguanidines readily reduced copper(II) to Cu(I) and were oxidized into a nitrosoamidine as previously observed in reactions performed with purified DBH. These data describe for the first time the reactivity of N-aryl-N′-hydroxyguanidines with a water-soluble copper(II) complex and help to understand the interaction of co-substrates with copper at the active site of DBH.     

Mutation in the substrate-binding site of aminopeptidase B confers new enzymatic properties

Aminopeptidase B (Ap-B) catalyzes the cleavage of arginine and lysine residues at the N-terminus of various peptide substrates. In vivo, it participates notably in the miniglucagon and cholecystokinin 8 processing, but the complete range of physiological functions of Ap-B remains to be discovered. Ap-B is a member of the M1 family of Zn²⁺-metallopeptidases that are characterized by two highly conserved motives, GXMEN (potential substrate binding site) and HEXXHX¹⁸E (Zn²⁺-binding site). In this study, mutagenesis and molecular modelling were used to investigate the enzymatic mechanism of Ap-B. Nineteen rat Ap-B mutants of the G₂₉₈XM₃₀₀E₃₀₁N₃₀₂ motif and one mutant of the HEIS₃₂₈HX¹⁸E motif were expressed in Escherichia coli. All mutations except G₂₉₈P, G₂₉₈S, and S₃₂₈A abolished the aminopeptidase activity. The S₃₂₈A mutant mimics the sequence of bovine Ap-B Zn²⁺-binding site, which differs from those of other mammalian Ap-B. This mutant conserved a canonical Ap-B activity. G₂₉₈S and G₂₉₈P mutants exhibit new enzymatic properties such as changes in their profile of inhibition and their sensitivity to Cl⁻ anions. Moreover, the G₂₉₈P mutant exhibits new substrate specificity. A structural analysis using circular dichroism, fluorescence spectroscopy, molecular modelling and dynamics was performed to investigate the role that residue G₂₉₈ plays in the catalytic mechanism of Ap-B. Our results show that G₂₉₈ is essential to Ap-B activity and participates to the substrate specificity of the enzyme.     

Characterisation of nitric oxide synthase activity in the tropical sea anemone Aiptasia pallida

The presence of nitric oxide synthase (EC 1.14.23 NOS) activity is demonstrated in the tropical marine cnidarian Aiptasia pallida (Verrill). Enzyme activity was assayed by measuring the conversion of [³H]arginine to [³H]citrulline. Optimal NOS activity was found to require NADPH. Activity was inhibited by the competitive NOS inhibitor N^G-methyl- -arginine ( -NMA), but not the arginase inhibitors -valine and -ornithine. NOS activity was predominantly cytosolic, and was characterised by a K_m for arginine of 19.05 μM and a V_max of 2.96 pmol/min per μg protein. Histochemical localisation of NOS activity using NADPH diaphorase staining showed the enzyme to be predominantly present in the epidermal cells and at the extremities of the mesoglea. These results provide a preliminary biochemical characterisation and histochemical localisation of NOS activity in A. pallida, an ecologically important sentinel species in tropical marine ecosystems.     

Characterization of Calmodulin-Free Murine Inducible Nitric-Oxide Synthase

Nitric-Oxide Synthase (NOS), that produces the biological signal molecule Nitric-Oxide (NO), exists in three different isoforms called, neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). All NOS isoforms require post-translational interaction with the calcium-binding protein, calmodulin (CaM) for manifesting their catalytic activity. However, CaM has been suggested to control the translational assembly of the enzyme as well, particularly in helping its inducible isoform, iNOS assume a stable, heme-replete, dimeric and active form. Expression of recombinant murine iNOS in E.coli in the absence of CaM has been previously shown to give extremely poor yield of the enzyme which was claimed to be absolutely heme-free, devoid of flavins, completely monomeric and catalytically inactive when compared to the heme-replete, active, dimeric iNOS, generated through co-expression with CaM. In contrast, we found that although iNOS expressed without CaM does produce significantly low amounts of the CaM-free enzyme, the iNOS thus produced, is not completely devoid of heme and is neither entirely monomeric nor absolutely bereft of catalytic activity as reported before. In fact, iNOS synthesized in the absence of CaM undergoes compromised heme incorporation resulting in extremely poor dimerization and activity compared to its counterpart co-expressed with CaM. Moreover, such CaM-free iNOS has similar flavin content and reductase activity as iNOS co-expressed with CaM, suggesting that CaM may not be as much required for the functional assembly of the iNOS reductase domain as its oxygenase domain. LC-MS/MS-based peptide mapping of the CaM-free iNOS confirmed that it had the same full-length sequence as the CaM-replete iNOS. Isothermal calorimetric measurements also revealed high affinity for CaM binding in the CaM-free iNOS and thus the possible presence of a CaM-binding domain. Thus CaM is essential but not indispensible for the assembly of iNOS and such CaM-free iNOS may help in elucidating the role of CaM on iNOS catalysis.