Reconstitution of pterin-free inducible nitric-oxide synthase

Inducible nitric-oxide synthase (NOS) was expressed and purified in the absence of 6(R)-tetrahydro-l-biopterin (H(4)B). Pterin-free NOS exhibits a Soret band (416-420 nm) characteristic of predominantly low spin heme and does not catalyze the formation of nitric oxide (. NO) (Rusche, K. M., Spiering, M. M., and Marletta, M. A. (1998) Biochemistry 37, 15503-15512). Reconstitution of pterin-free NOS with H(4)B was monitored by a shift in the Soret band to 396-400 nm, the recovery of.NO-forming activity, and the measurement of H(4)B bound to the enzyme. As assessed by these properties, H(4)B binding was not rapid and required the presence of a reduced thiol. Spectral changes and recovery of activity were incomplete in the absence of reduced thiol. Full reconstitution of holoenzyme activity and stoichiometric H(4)B binding was achieved in the presence of 5 mm glutathione (GSH). Preincubation with GSH before the addition of H(4)B decreased, whereas lower concentrations of GSH extended, the time required for reconstitution. Six protected cysteine residues in pterin-free NOS were identified by labeling of NOS with cysteine-directed reagents before and after reduction with GSH. Heme and metal content of pterin-free and H(4)B-reconstituted NOS were also measured and were found to be independent of H(4)B content. Additionally, pterin-free NOS was reconstituted with 6-methylpterin analogs, including redox-stable deazapterins. Reconstitution with the redox-stable pterin analogs was neither time- nor thiol-dependent. Apparent binding constants were determined for the 6-methyl- (50 microm) and 6-ethoxymethyl (200 microm) deazapterins. The redox-stable pterin analogs appear to bind to NOS in a different manner than H(4)B.     

Regulation of the monomer-dimer equilibrium in inducible nitric-oxide synthase by nitric oxide

The oxygenase domain of inducible nitric-oxide synthase exists as a functional tight homodimer in the presence of the substrate L-arginine and the cofactor tetrahydrobiopterin (H4B). In the absence of H4B, the enzyme is a mixture of monomer and loose dimer. We show that exposure of H4B-free enzyme to NO induces dissociation of the loose dimer into monomers in a reaction that follows single exponential decay kinetics with a lifetime of approximately 300 min. It is followed by a faster autoreduction reaction of the heme iron with a lifetime of approximately 30 min and the concurrent breakage of the proximal iron-thiolate bond, forming a five-coordinate NO-bound ferrous species. Mass spectrometry revealed that the NO-induced monomerization is associated with intramolecular disulfide bond formation between Cys104 and Cys109, located in the zinc-binding motif. The regulatory effect of NO as a dimer inhibitor is discussed in the context of the structure/function relationships of this enzyme.     

Mutational analysis of the tetrahydrobiopterin-binding site in inducible nitric-oxide synthase.

Inducible nitric-oxide synthase (iNOS) is a hemeprotein that requires tetrahydrobiopterin (H4B) for activity. The influence of H4B on iNOS structure-function is complex, and its exact role in nitric oxide (NO) synthesis is unknown. Crystal structures of the mouse iNOS oxygenase domain (iNOSox) revealed a unique H4B-binding site with a high degree of aromatic character located in the dimer interface and near the heme. Four conserved residues (Arg-375, Trp-455, Trp-457, and Phe-470) engage in hydrogen bonding or aromatic stacking interactions with the H4B ring. We utilized point mutagenesis to investigate how each residue modulates H4B function. All mutants contained heme ligated to Cys-194 indicating no deleterious effect on general protein structure. Ala mutants were monomers except for W457A and did not form a homodimer with excess H4B and Arg. However, they did form heterodimers when paired with a full-length iNOS subunit, and these were either fully or partially active regarding NO synthesis, indicating that preserving residue identities or aromatic character is not essential for H4B binding or activity. Aromatic substitution at Trp-455 or Trp-457 generated monomers that could dimerize with H4B and Arg. These mutants bound Arg and H4B with near normal affinity, but Arg could not displace heme-bound imidazole, and they had NO synthesis activities lower than wild-type in both homodimeric and heterodimeric settings. Aromatic substitution at Phe-470 had no significant effects. Together, our work shows how hydrogen bonding and aromatic stacking interactions of Arg-375, Trp-457, Trp-455, and Phe-470 influence iNOSox dimeric structure, heme environment, and NO synthesis and thus help modulate the multiple effects of H4B.     

Dynamic receptor-dependent activation of inducible nitric-oxide synthase by ERK-mediated phosphorylation of Ser745

Nitric oxide (NO) is a pleiotropic regulator of vascular function, and its overproduction by inducible nitric-oxide synthase (iNOS) in inflammatory conditions plays an important role in the pathogenesis of vascular diseases. iNOS activity is thought to be regulated primarily at the level of expression to generate "high output" NO compared with constitutive NO synthases. Here we show iNOS activity is acutely up-regulated by activation of the B1-kinin receptor (B1R) in human endothelial cells or transfected HEK293 cells to generate 2.5-5-fold higher NO than that stimulated by Arg alone. Increased iNOS activity was dependent on B1R activation of the MAPK ERK. In HEK293 cells transfected with human iNOS and B1R, ERK phosphorylated iNOS on Ser745 as determined by Western analysis using phospho-Ser antibody, in vitro kinase assays with activated ERK, and MALDI-TOF mass spectrometry. Mutation of Ser745 to Ala did not affect basal iNOS activity but eliminated iNOS phosphorylation and activation in response to B1R agonist. Mutation of Ser745 to Asp resulted in a basally hyperactive iNOS whose activity was not further increased by B1R agonist. ERK and phospho-ERK (after B1R activation) were co-localized with iNOS as determined by confocal fluorescence microscopy. Furthermore, ERK co-immunoprecipitated with iNOS. The discovery that iNOS can be phosphorylated by ERK and acutely activated by receptor-mediated signaling reveals a new level of regulation for this isoform. These findings provide a novel therapeutic target to explore in the treatment of vascular inflammatory diseases.     

Regulation of inducible nitric-oxide synthase by the SPRY domain- and SOCS box-containing proteins

Inducible nitric-oxide synthase (iNOS, NOS2) plays a prominent role in macrophage bactericidal and tumoricidal activities. A relatively large amount of NO produced via iNOS, however, also targets the macrophage itself for apoptotic cell death. To uncover the intrinsic mechanisms of iNOS regulation, we have characterized the SPRY domain- and SOCS box-containing protein 1 (SPSB1), SPSB2, and SPSB4 that interact with the N-terminal region of iNOS in a D-I-N-N-N sequence-dependent manner. Fluorescence microscopy revealed that these SPSB proteins can induce the subcellular redistribution of iNOS from dense regions to diffused expression in a SOCS box-dependent manner. In immunoprecipitation studies, both Elongin C and Cullin-5, components of the multi-subunit E3 ubiquitin ligase, were found to bind to iNOS via SPSB1, SPSB2, or SPSB4. Consistently, iNOS was polyubiquitinated and degraded in a proteasome-dependent manner when SPSB1, SPSB2, or SPSB4 was expressed. SPSB1 and SPSB4 had a greater effect on iNOS regulation than SPSB2. The iNOS N-terminal fragment (residues 1-124 of human iNOS) could disrupt iNOS-SPSB interactions and inhibit iNOS degradation. In lipopolysaccharide-treated macrophages, this fragment attenuated iNOS ubiquitination and substantially prolonged iNOS lifetime, resulting in a corresponding increase in NO production and enhanced NO-dependent cell death. These results not only demonstrate the mechanism of SPSB-mediated iNOS degradation and the relative contributions of different SPSB proteins to iNOS regulation, but also show that iNOS levels are sophisticatedly regulated by SPSB proteins in activated macrophages to prevent overproduction of NO that could trigger detrimental effects, such as cytotoxicity.     

Heme insertion, assembly, and activation of apo-neuronal nitric-oxide synthase in vitro

It has been established that in the case of inducible NO synthase (NOS), a functionally active homodimer is assembled from the heme-deficient monomeric apo-NOS in vitro by the addition of heme, whereas the heme-deficient neuronal isoform (apo-nNOS) is at best only partially activated. In the current study we have discovered that reactive oxygen species, which can be removed by the addition of superoxide dismutase and catalase, destroy the heme and limit the activation of apo-nNOS in vitro. With the use of these improved conditions, we show for the first time that heme insertion is a rapid process that results in formation of a heme-bound monomeric nNOS that is able to form the ferrous-CO P450 complex but is unable to synthesize NO. A slow process requiring more than 90 min is required for dimerization and activation of this P450 intermediate to give an enzyme with a specific activity of approximately 1100 nmol of NO formed/min/mg of protein, similar to that of the native enzyme. Interestingly, the dimer is not SDS-resistant and is not the same dimer that forms in vivo. These studies indicate at least two intermediates in the assembly of nNOS and advance our understanding of the regulation of nNOS.     

Endothelial heme oxygenase-1 induction by hypoxia. Modulation by inducible nitric-oxide synthase and S-nitrosothiols

The stress protein heme oxygenase-1 (HO-1) is induced in endothelial cells exposed to nitric oxide (NO)-releasing agents, and this process is finely modulated by thiols (Foresti, R., Clark, J. E., Green, C. J., and Motterlini R. (1997) J. Biol. Chem. 272, 18411-18417). Here, we report that up-regulation of HO-1 in aortic endothelial cells by severe hypoxic conditions (pO(2) 相似文献   

Activation of peroxynitrite by inducible nitric-oxide synthase: a direct source of nitrative stress

In mammals, nitric oxide (NO) is an essential biological mediator that is exclusively synthesized by nitric-oxide synthases (NOSs). However, NOSs are also directly or indirectly responsible for the production of peroxynitrite, a well known cytotoxic agent involved in numerous pathophysiological processes. Peroxynitrite reactivity is extremely intricate and highly depends on activators such as hemoproteins. NOSs present, therefore, the unique ability to both produce and activate peroxynitrite, which confers upon them a major role in the control of peroxynitrite bioactivity. We report here the first kinetic analysis of the interaction between peroxynitrite and the oxygenase domain of inducible NOS (iNOSoxy). iNOSoxy binds peroxynitrite and accelerates its decomposition with a second order rate constant of 22 x 10(4) m(-1)s(-1) at pH 7.4. This reaction is pH-dependent and is abolished by the binding of substrate or product. Peroxynitrite activation is correlated with the observation of a new iNOS heme intermediate with specific absorption at 445 nm. iNOSoxy modifies peroxynitrite reactivity and directs it toward one-electron processes such as nitration or one-electron oxidation. Taken together our results suggest that, upon binding to iNOSoxy, peroxynitrite undergoes homolytic cleavage with build-up of an oxo-ferryl intermediate and concomitant release of a NO(2)(.) radical. Successive cycles of peroxynitrite activation were shown to lead to iNOSoxy autocatalytic nitration and inhibition. The balance between peroxynitrite activation and self-inhibition of iNOSoxy may determine the contribution of NOSs to cellular oxidative stress.     

Stabilization and characterization of a heme-oxy reaction intermediate in inducible nitric-oxide synthase

Nitric-oxide synthases (NOS) are heme-thiolate enzymes that N-hydroxylate L-arginine (L-Arg) to make NO. NOS contain a unique Trp residue whose side chain stacks with the heme and hydrogen bonds with the heme thiolate. To understand its importance we substituted His for Trp188 in the inducible NOS oxygenase domain (iNOSoxy) and characterized enzyme spectral, thermodynamic, structural, kinetic, and catalytic properties. The W188H mutation had relatively small effects on l-Arg binding and on enzyme heme-CO and heme-NO absorbance spectra, but increased the heme midpoint potential by 88 mV relative to wild-type iNOSoxy, indicating it decreased heme-thiolate electronegativity. The protein crystal structure showed that the His188 imidazole still stacked with the heme and was positioned to hydrogen bond with the heme thiolate. Analysis of a single turnover L-Arg hydroxylation reaction revealed that a new heme species formed during the reaction. Its build up coincided kinetically with the disappearance of the enzyme heme-dioxy species and with the formation of a tetrahydrobiopterin (H4B) radical in the enzyme, whereas its subsequent disappearance coincided with the rate of l-Arg hydroxylation and formation of ferric enzyme. We conclude: (i) W188H iNOSoxy stabilizes a heme-oxy species that forms upon reduction of the heme-dioxy species by H4B. (ii) The W188H mutation hinders either the processing or reactivity of the heme-oxy species and makes these steps become rate-limiting for l-Arg hydroxylation. Thus, the conserved Trp residue in NOS may facilitate formation and/or reactivity of the ultimate hydroxylating species by tuning heme-thiolate electronegativity.     

Surface charge interactions of the FMN module govern catalysis by nitric-oxide synthase

The FMN module of nitric-oxide synthase (NOS) plays a pivotal role by transferring NADPH-derived electrons to the enzyme heme for use in oxygen activation. The process may involve a swinging mechanism in which the same face of the FMN module accepts and provides electrons during catalysis. Crystal structure shows that this face of the FMN module is electronegative, whereas the complementary interacting surface is electropositive, implying that charge interactions enable function. We used site-directed mutagenesis to investigate the roles of six electronegative surface residues of the FMN module in electron transfer and catalysis in neuronal NOS. Results are interpreted in light of crystal structures of NOS and related flavoproteins. Neutralizing or reversing the negative charge of each residue altered the NO synthesis, NADPH oxidase, and cytochrome c reductase activities of neuronal NOS and also altered heme reduction. The largest effects occurred at the NOS-specific charged residue Glu(762). Together, the results suggest that electrostatic interactions of the FMN module help to regulate electron transfer and to minimize flavin autoxidation and the generation of reactive oxygen species during NOS catalysis.     

Mechanistic studies on the intramolecular one-electron transfer between the two flavins in the human neuronal nitric-oxide synthase and inducible nitric-oxide synthase flavin domains

Neuronal nitric-oxide synthase (nNOS) differs from inducible NOS (iNOS) in both its dependence on the intracellular Ca2+ concentration and the production rate of NO. To investigate what difference(s) exist between the two NOS flavin domains at the electron transfer level, we isolated the recombinant human NOS flavin domains, which were co-expressed with human calmodulin (CaM). The flavin semiquinones, FADH* and FMNH*, in both NOSs participate in the regulation of one-electron transfer within the flavin domain. Each semiquinone can be identified by a characteristic absorption peak at 520 nm (Guan, Z.-W., and Iyanagi, T. (2003) Arch. Biochem. Biophys. 412, 65-76). NADPH reduction of the FAD and FMN redox centers by the CaM-bound flavin domains was studied by stopped-flow and rapid scan spectrometry. Reduction of the air-stable semiquinone (FAD-FMNH*) of both domains with NADPH showed that the extent of conversion of FADH2/FMNH* to FADH*/FMNH2 in the iNOS flavin domain was greater than that of the nNOS flavin domain. The reduction of both oxidized domains (FAD-FMN) with NADPH resulted in the initial formation of a small amount of disemiquinone, which then decayed. The rate of intramolecular electron transfer between the two flavins in the iNOS flavin domain was faster than that of the nNOS flavin domain. In addition, the formation of a mixture of the two- and four-electron-reduced states in the presence of excess NADPH was different for the two NOS flavin domains. The data indicate a more favorable formation of the active intermediate FMNH2 in the iNOS flavin domain.     

Regulation of prostaglandin biosynthesis by nitric oxide is revealed by targeted deletion of inducible nitric-oxide synthase

We investigated the effects of targeted deletion of the inducible NO synthase (iNOS) gene on the formation of prostaglandins in vivo and ex vivo. Peritoneal macrophages were obtained from control and iNOS-deficient mice, and prostaglandin E(2) (PGE(2)) was quantified after stimulation with gamma-interferon and lipopolysaccharide to induce COX-2. Total nitrate and nitrite production was completely abolished in cells from iNOS-deficient animals compared with control cells. PGE(2) formation by cells from iNOS-deficient animals was decreased compared with cells from control animals 80% at 12 h (0.85 +/- 0.90 ng/10(6) cells versus 15.4 +/- 2.1 ng/10(6) cells, p < 0.01) and 74% at 24 h (9.4 +/- 4.3 ng/10(6) cells versus 36.8 +/- 4.1 ng/10(6) cells, p < 0.01). COX-2 protein expression was not significantly different in cells from control or knockout animals. Levels of PGE(2) in the urine of iNOS-deficient mice were decreased 78% (0.24 +/- 0.14 ng/mg of creatinine versus 1.09 +/- 0.66 ng/mg of creatinine, p < 0.01) compared with control animals. In addition, the levels of urinary F(2)-isoprostanes, an index of endogenous oxidant stress, were significantly decreased in iNOS-deficient animals. In contrast, the levels of thromboxane B(2) derived from platelets allowed to aggregate ex vivo were significantly increased in iNOS-deficient mice compared with wild-type mice. These studies support the hypothesis that NO and/or NO-derived species modulate cyclooxygenase activity and eicosanoid production in vivo.     

Mechanistic studies with potent and selective inducible nitric-oxide synthase dimerization inhibitors.

A series of potent and selective inducible nitric-oxide synthase (iNOS) inhibitors was shown to prevent iNOS dimerization in cells and inhibit iNOS in vivo. These inhibitors are now shown to block dimerization of purified human iNOS monomers. A 3H-labeled inhibitor bound to full-length human iNOS monomer with apparent Kd approximately 1.8 nm and had a slow off rate, 1.2 x 10(-4) x s(-1). Inhibitors also bound with high affinity to both murine full-length and murine oxygenase domain iNOS monomers. Spectroscopy and competition binding with imidazole confirmed an inhibitor-heme interaction. Inhibitor affinity in the binding assay (apparent Kd values from 330 pm to 27 nm) correlated with potency in a cell-based iNOS assay (IC50 values from 290 pm to 270 nm). Inhibitor potency in cells was not prevented by medium supplementation with l-arginine or sepiapterin, but inhibition decreased with time of addition after cytokine stimulation. The results are consistent with a mechanism whereby inhibitors bind to a heme-containing iNOS monomer species to form an inactive iNOS monomer-heme-inhibitor complex in a pterin- and l-arginine-independent manner. The selectivity for inhibiting dimerization of iNOS versus endothelial and neuronal NOS suggests that the energetics and kinetics of monomer-dimer equilibria are substantially different for the mammalian NOS isoforms. These inhibitors provide new research tools to explore these processes.     

Dynamic regulation of the inducible nitric-oxide synthase by NO: comparison with the endothelial isoform

We studied by ultrafast time-resolved absorption spectroscopy the geminate recombination of NO to the oxygenase domain of the inducible NO synthase, iNOSoxy, and to mutated proteins at position Trp-457. This tryptophan interacts with the tetrahydrobiopterin cofactor BH4, and W457A/F mutations largely reduced the catalytic formation of NO. BH4 decreases the rate of NO rebinding to the ferric iNOSoxy compared with that measured in its absence. The pterin has a larger effect on W457A/F than on the WT protein by increasing NO release from the protein. Therefore, BH4 raises the energy barrier for NO recombination to the mutated proteins in contrast with our observations on eNOS (Slama-Schwok, A., Négrerie, M., Berka, V., Lambry, J.-C., Tsai, A.-L., Vos, M., and Martin, J.-L. (2002) J. Biol. Chem. 277, 7581-7586). Thus, we show a differential effect of BH4 on NO release from eNOS and iNOS. Compared with the position of this residue in the BH4-repleted enzyme, simulations of the NO dissociation dynamics point out at a swing of Trp-457 toward the missing pterin in the absence of BH4. NO geminate-rebinding data show a more efficient NO release from eNOS than from iNOS once NO is formed. Consistently, NO produced by iNOS is regulated by its ferric nitrosyl complex in contrast with eNOS. We show that the small enhancement of the NO geminate recombination rate in W457A/F compared with that in the WT enzyme cannot explain the decrease of NO yield because of the mutation; the major effect of the mutation thus arises from an uncoupled catalysis (Wang, Z. Q., Wei, C. C., Ghosh, S., Meade, A. L., Hemann, C., Hille, R., and Stuehr, D. J. (2001) Biochemistry 40, 12819-12825).     

Substrate-specific interactions with the heme-bound oxygen molecule of nitric-oxide synthase

We report the characterization by resonance Raman spectroscopy of the oxygenated complex (Fe(II)O(2)) of nitric-oxide synthases of Staphylococcus aureus (saNOS) and Bacillus subtilis (bsNOS) saturated with N(omega)-hydroxy-l-arginine. The frequencies of the nu(Fe-O) and nu(O-O) modes were 530 and 1135 cm(-), respectively, in both the presence and absence of tetrahydrobiopterin. On the basis of a comparison of these frequencies with those of saNOS and bsNOS saturated with l-arginine (nu(Fe-O) at 517 cm(-1) and nu(O-O) at 1123 cm(-1)) and those of substrate-free saNOS (nu(Fe-O) at 517 and nu(O-O) at 1135 cm(-1)) (Chartier, F. J. M., Blais, S. P., and Couture, M. (2006) J. Biol. Chem. 281, 9953-9962), we propose two models that account for the frequency shift of nu(Fe-O) (but not nu(O-O)) upon N(omega)-hydroxy-l-arginine binding as well as the frequency shift of nu(O-O) (but not nu(Fe-O)) upon l-arginine binding. The implications of these substrate-specific interactions with respect to catalysis by NOSs are discussed.