Crystal structure of nitric oxide synthase bound to nitro indazole reveals a novel inactivation mechanism.

Nitric oxide is generated under normal and pathophysiological conditions by three distinct isoforms of nitric oxide synthase (NOS). A small-molecule inhibitor of NOS (3-Br-7-nitroindazole, 7-NIBr) is profoundly neuroprotective in mouse models of stroke and Parkinson's disease. We report the crystal structure of the catalytic heme domain of endothelial NOS complexed with 7-NIBr at 1.65 A resolution. Critical to the binding of 7-NIBr at the substrate site is the adoption by eNOS of an altered conformation, in which a key glutamate residue swings out toward one of the heme propionate groups. Perturbation of the heme propionate ensues and eliminates the cofactor tetrahydrobiopterin-heme interaction. We also present three crystal structures that reveal how alterations at the substrate site facilitate 7-NIBr and structurally dissimilar ligands to occupy the cofactor site.     

Alternative pathways of nitric oxide formation in lactobacilli: EPR evidence for nitric oxide synthase activity

The study of the ability of Lactobacillus plantarum 8P-A3 to synthesize nitric oxide (NO) showed that this strain lacks nitrite reductase. However, analysis by the EPR method revealed the presence of nitric oxide synthase activity in this strain. Like mammalian nitric oxide synthase, lactobacillar NO synthase is involved in the formation of nitric oxide from L-arginine. L. plantarum 8P-A3 does not produce NO in the course of denitrification process. The regulatory role of NO in symbiotic bacteria is discussed.     

Structures of the neuronal and endothelial nitric oxide synthase heme domain with D-nitroarginine-containing dipeptide inhibitors bound

In a continuing effort to unravel the structural basis for isoform-selective inhibition of nitric oxide synthase (NOS) by various inhibitors, we have determined the crystal structures of the nNOS and eNOS heme domain bound with two D-nitroarginine-containing dipeptide inhibitors, D-Lys-D-Arg(NO)2-NH(2) and D-Phe-D-Arg(NO)2-NH(2). These two dipeptide inhibitors exhibit similar binding modes in the two constitutive NOS isozymes, which is consistent with the similar binding affinities for the two isoforms as determined by K(i) measurements. The D-nitroarginine-containing dipeptide inhibitors are not distinguished by the amino acid difference between nNOS and eNOS (Asp 597 and Asn 368, respectively) which is key in controlling isoform selection for nNOS over eNOS observed for the L-nitroarginine-containing dipeptide inhibitors reported previously [Flinspach, M., et al. (2004) Nat. Struct. Mol. Biol. 11, 54-59]. The lack of a free alpha-amino group on the D-nitroarginine moiety makes the dipeptide inhibitor steer away from the amino acid binding pocket near the active site. This allows the inhibitor to extend into the solvent-accessible channel farther away from the active site, which enables the inhibitors to explore new isoform-specific enzyme-inhibitor interactions. This might be the structural basis for why these D-nitroarginine-containing inhibitors are selective for nNOS (or eNOS) over iNOS.     

Chronic nitric oxide synthase blockade desensitizes the heart to the negative metabolic effects of nitric oxide

The consequences of chronic nitric oxide synthase (NOS) blockade on the myocardial metabolic and guanylyl cyclase stimulatory effects of exogenous nitric oxide (NO) were determined. Thirty-three anesthetized open-chest rabbits were randomized into four groups: control, NO donor S-nitroso-N-acetyl-penicillamine (SNAP, 10(-4 )M), NOS blocking agent N(G)-nitro-L-arginine methyl ester (L-NAME, 20 mg/kg/day) for 10 days followed by a 24 hour washout and L-NAME for 10 days followed by a 24 hour washout plus SNAP. Myocardial O(2) consumption was determined from coronary flow (microspheres) and O(2) extraction (microspectrophotometry). Cyclic GMP and guanylyl cyclase activity were determined by radioimmunoassay. There were no baseline metabolic, functional or hemodynamic differences between control and L-NAME treated rabbits. SNAP in controls caused a reduction in O(2) consumption (SNAP 5.9+/-0.6 vs. control 8.4+/-0.8 ml O(2)/min/100 g) and a rise in cyclic GMP (SNAP 18.3+/-3.8 vs. control 10.4+/-0.9 pmol/g). After chronic L-NAME treatment, SNAP caused no significant changes in O(2) consumption (SNAP 7.1+/-0.8 vs. control 6.4+/-0.7) or cyclic GMP (SNAP 14.2+/-1.8 vs. control 12.1+/-1.3). In controls, guanylyl cyclase activity was significantly stimulated by SNAP (216.7+/-20.0 SNAP vs. 34.4+/-2.5 pmol/mg/min base), while this increase was blunted after L-NAME (115.9+/-24.5 SNAP vs. 24.9+/-4.7 base). These results demonstrated that chronic NOS blockade followed by washout blunts the response to exogenous NO, with little effect on cyclic GMP or myocardial O(2) consumption. This was related to reduced guanylyl cyclase activity after chronic L-NAME. These results suggest that, unlike many receptor systems, the NO-cyclic GMP signal transduction system becomes downregulated upon chronic inhibition.     

Histochemical methods for detecting nitric oxide synthase

Summary The three isoforms of nitric oxide synthase (NOS), neuronal (nNOS), endothelial (eNOS), and inducible (iNOS), can be visualized in cells and tissues by NADPH-diaphorase (NADPH-d) histochemistry, immunocytochemistry and in situ hybridization. Histochemical demonstration of NADPH-d shows the formazan final reaction product as a solid blue deposit. The ultrastructural localization of NADPH-d in the rat hippocampus showed an electron-dense deposit on membranes predominantly of the endoplasmic reticulum. The immunohistochemical demonstration of nNOS, using the nickel enhancement technique, shows positive reaction product over the dendrites and the soma of the nerve cell in the rat brain. Ultrastructural localization of nNOS in whole mount preparations of myenteric plexus and circular smooth muscle from guinea-pig ileum shows that NOS immunoreactivity was patchily distributed in myenteric neurones and was not specifically associated with any intracellular organelles or with plasma membranes. In situ hybridization, using radio-labelled probes, was used to study nNOS mRNA in lumbar dorsal root ganglia after peripheral transection of the sciatic nerve in rats. Labelling of the NOS mRNA-positive neurones is observed as a series of dense granules over the entire cell. NADPH-d histochemistry, immunocytochemistry and in situ hybridization each have a significant role to play in the localization of NOS. NADPH-d detects an enzyme associated with the NOS molecule, immunocytochemistry detects the NOS molecule, and in situ hybridization detects mRNA for NOS. Therefore, if each of these techniques is applied in carefully controlled experiments, consideration of the accumulated data should be valuable in revealing insights into the biology of NOS.     

Structural basis for endothelial nitric oxide synthase binding to calmodulin

The enzyme nitric oxide synthase (NOS) is exquisitely regulated in vivo by the Ca(2+) sensor protein calmodulin (CaM) to control production of NO, a key signaling molecule and cytotoxin. The differential activation of NOS isozymes by CaM has remained enigmatic, despite extensive research. Here, the crystallographic structure of Ca(2+)-loaded CaM bound to a 20 residue peptide comprising the endothelial NOS (eNOS) CaM-binding region establishes their individual conformations and intermolecular interactions, and suggests the basis for isozyme-specific differences. The alpha-helical eNOS peptide binds in an antiparallel orientation to CaM through extensive hydrophobic interactions. Unique NOS interactions occur with: (i). the CaM flexible central linker, explaining its importance in NOS activation; and (ii). the CaM C-terminus, explaining the NOS-specific requirement for a bulky, hydrophobic residue at position 144. This binding mode expands mechanisms for CaM-mediated activation, explains eNOS deactivation by Thr495 phosphorylation, and implicates specific hydrophobic residues in the Ca(2+) independence of inducible NOS.     

昆虫一氧化氮及其合酶的研究进展

一氧化氮作为一种重要的信息分子 ,参与调节昆虫嗅觉、视觉、机械感受、发育、机体防御及学习行为。该文从生理、生化、形态定位以及信号转导几方面综述了有关昆虫一氧化氮及其合酶的最新研究进展。     

Neuronal nitric oxide synthase contributes to the regulation of hematopoiesis

Nitric oxide (NO) signaling is important for the regulation of hematopoiesis. However, the role of individual NO synthase (NOS) isoforms is unclear. Our results indicate that the neuronal NOS isoform (nNOS) regulates hematopoiesis in vitro and in vivo. nNOS is expressed in adult bone marrow and fetal liver and is enriched in stromal cells. There is a strong correlation between expression of nNOS in a panel of stromal cell lines established from bone marrow and fetal liver and the ability of these cell lines to support hematopoietic stem cells; furthermore, NO donor can further increase this ability. The number of colonies generated in vitro from the bone marrow and spleen of nNOS-null mutants is increased relative to wild-type or inducible- or endothelial NOS knockout mice. These results describe a new role for nNOS beyond its action in the brain and muscle and suggest a model where nNOS, expressed in stromal cells, produces NO which acts as a paracrine regulator of hematopoietic stem cells.     

Electron paramagnetic resonance characterization of tetrahydrobiopterin radical formation in bacterial nitric oxide synthase compared to mammalian nitric oxide synthase

H(4)B is an essential catalytic cofactor of the mNOSs. It acts as an electron donor and activates the ferrous heme-oxygen complex intermediate during Arg oxidation (first step) and NOHA oxidation (second step) leading to nitric oxide and citrulline as final products. However, its role as a proton donor is still debated. Furthermore, its exact involvement has never been explored for other NOSs such as NOS-like proteins from bacteria. This article proposes a comparative study of the role of H(4)B between iNOS and bsNOS. In this work, we have used freeze-quench to stop the arginine and NOHA oxidation reactions and trap reaction intermediates. We have characterized these intermediates using multifrequency electron paramagnetic resonance. For the first time, to our knowledge, we report a radical formation for a nonmammalian NOS. The results indicate that bsNOS, like iNOS, has the capacity to generate a pterin radical during Arg oxidation. Our current electron paramagnetic resonance data suggest that this radical is protonated indicating that H(4)B may not transfer any proton. In the 2nd step, the radical trapped for iNOS is also suggested to be protonated as in the 1st step, whereas it was not possible to trap a radical for the bsNOS 2nd step. Our data highlight potential differences for the catalytic mechanism of NOHA oxidation between mammalian and bacterial NOSs.     

Binding of CAP70 to inducible nitric oxide synthase and implications for the vectorial release of nitric oxide in polarized cells

In this article we analyze the mechanisms by which the C-terminal four amino acids of inducible nitric oxide synthase (NOS2) interact with proteins that contain PDZ (PSD-95/DLG/ZO-1) domains resulting in the translocation of NOS2 to the cellular apical domain. It has been reported that human hepatic NOS2 associates to EBP50, a protein with two PDZ domains present in epithelial cells. We describe herein that NOS2 binds through its four carboxy-terminal residues to CAP70, a protein that contains four PDZ modules that is targeted to apical membranes. Interestingly, this interaction augments both the cytochrome c reductase and .NO-synthase activities of NOS2. Binding of CAP70 to NOS2 also results in an increase in the population of active NOS2 dimers. In addition, CAP70 participates in the correct subcellular targeting of NOS2 in a process that is also dependent on the acylation state of the N-terminal end of NOS2. Hence, nonpalmitoylated NOS2 is unable to progress toward the apical side of the cell despite its interaction with either EBP50 or CAP70. Likewise, if we abrogate the interaction of NOS2 with either EBP50 or CAP70 by fusing the GFP reporter to the carboxy-terminal end of NOS2 palmitoylation is not sufficient to confer an apical targeting.     

ENDOR spectroscopic studies of stable semiquinone radicals bound to the Escherichia coli cytochrome bo3 quinol oxidase.

The putative oxidation of ubiquinol by the cytochrome bo3 terminal oxidase of Escherichia coli in sequential one-electron steps requires stabilization of the semiquinone. ENDOR spectroscopy has recently been used to study the native ubisemiquinone radical formed in the cytochrome bo3 quinone-binding site [Veselov, A.V., Osborne, J.P., Gennis, R.B. & Scholes, C.P. (2000) Biochemistry 39, 3169-3175]. Comparison of these spectra with those from the decyl-ubisemiquinone radical in vitro indicated that the protein induced large changes in the electronic structure of the ubisemiquinone radical. We have used quinone-substitution experiments to obtain ENDOR spectra of ubisemiquinone, phyllosemiquinone and plastosemiquinone anion radicals bound at the cytochrome bo3 quinone-binding site. Large changes in the electronic structures of these semiquinone anion radicals are induced on binding to the cytochrome bo3 oxidase. The changes in electronic structure are, however, independent of the electronic structures of these semiquinones in vitro. Thus it is shown to be the structure of this binding site in the protein, not the covalent structure of the bound quinone, that determines the electronic structure of the protein-bound semiquinone.     

Kinetic studies on the successive reaction of neuronal nitric oxide synthase from L-arginine to nitric oxide and L-citrulline

Rat neuronal nitric oxide synthase (nNOS) was heterologously expressed in Escherichia coliand purified. The conversion of L-arginine to N(omega)-hydroxy-L-arginine and further to L-citrulline in one cycle of the reaction of the purified nNOS was measured with the reaction rapid quenching method using (3)H-L-arginine as the substrate. It was found that most of the produced (3)H-N(omega)-hydroxy-L-arginine was successively hydroxylated to (3)H-L-citrulline without leaving the enzyme. From the analysis of time courses, the rate constants for each reaction step, and also for the dissociation of the intermediate, were estimated at various temperature in which the rates for the first and the second reactions were not much different each other but the rate for the dissociation of (3)H-N(omega)-hydroxy-L-arginine from the enzyme was significantly slow. Under the steady-state reaction condition, almost all of the nNOS was estimated to be active from the amount of burst formation of L-citrulline in the pre-steady state. The rate constant for the dissociation of the product L-citrulline from nNOS was calculated from the combination of results of the rapid quenching experiments and the metabolism of L-arginine in the presence of an excess amount of substrate, which was the smallest among all the rate constants in one cycle of the nNOS reaction. The activation energies for all the reaction steps were determined from the temperature dependence of the rate constants, which revealed that the rate-determining step of the nNOS reaction in the steady state was the dissociation of the product L-citrulline from the enzyme.     

Genomic structure of nitric oxide synthase in the terrestrial slug is highly conserved

Nitric oxide (NO) is a key molecule in olfactory information processing across animal species. To gain insight into the genetic basis of NO generation, we investigated the genomic structure of nitric oxide synthase (NOS) in the terrestrial slug Limax because slugs use olfaction as their primary food detection system. The full length cDNA of limNOS encodes a protein consisting of 1632 amino acids that has a PSD-95/discs-large/ZO-1 (PDZ) domain in its N-terminus and 6 other cofactor-binding domains. The limNOS gene consists of 33 exons and spans at least 107 kb of the genome. Almost all the exon-intron boundaries are conserved in Limax and human nNOS and the organization of the Limax genome is more similar to that of humans than to Drosophila, indicating that there was an accelerated evolution of the Drosophila genome during evolution. These results imply that there was a highly conservative selective pressure imposed on NOS gene structure during the evolution of mollusks and vertebrates.     

Nitric oxide and nitric oxide synthase activity in plants

Research on NO in plants has gained considerable attention in recent years mainly due to its function in plant growth and development and as a key signalling molecule in different intracellular processes in plants. The NO emission from plants is known since the 1970s, and now there is abundant information on the multiple effects of exogenously applied NO on different physiological and biochemical processes of plants. The physiological function of NO in plants mainly involves the induction of different processes, including the expression of defence-related genes against pathogens and apoptosis/programmed cell death (PCD), maturation and senescence, stomatal closure, seed germination, root development and the induction of ethylene emission. NO can be produced in plants by non-enzymatic and enzymatic systems. The NO-producing enzymes identified in plants are nitrate reductase, and several nitric oxide synthase-like activities, including one localized in peroxisomes which has been biochemically characterized. Recently, two genes of plant proteins with NOS activity have been isolated and characterized for the first time, and both proteins do not have sequence similarities to any mammalian NOS isoform. However, different evidence available indicate that there are other potential enzymatic sources of NO in plants, including xanthine oxidoreductase, peroxidase, cytochrome P450, and some hemeproteins. In plants, the enzymatic production of the signal molecule NO, either constitutive or induced by different biotic/abiotic stresses, may be a much more common event than was initially thought.     

Rate of nitric oxide scavenging by hemoglobin bound to haptoglobin

Cell-free hemoglobin, released from the red cell, may play a major role in regulating the bioavailability of nitric oxide. The abundant serum protein haptoglobin, rapidly binds to free hemoglobin forming a stable complex accelerating its clearance. The haptoglobin gene is polymorphic with two classes of alleles denoted 1 and 2. We have previously demonstrated that the haptoglobin 1 protein–hemoglobin complex is cleared twice as fast as the haptoglobin 2 protein–hemoglobin complex. In this report, we explored whether haptoglobin binding to hemoglobin reduces the rate of nitric oxide scavenging using time-resolved absorption spectroscopy. We found that both the haptoglobin 1 and haptoglobin 2 protein complexes react with nitric oxide at the same rate as unbound cell-free hemoglobin. To confirm these results we developed a novel assay where free hemoglobin and hemoglobin bound to haptoglobin competed in the reaction with NO. The relative rate of the NO reaction was then determined by examining the amount of reacted species using analytical ultracentrifugation. Since complexation of hemoglobin with haptoglobin does not reduce NO scavenging, we propose that the haptoglobin genotype may influence nitric oxide bioavailability by determining the clearance rate of the haptoglobin–hemoglobin complex. We provide computer simulations showing that a twofold difference in the rate of uptake of the haptoglobin–hemoglobin complex by macrophages significantly affects nitric oxide bioavailability thereby providing a plausible explanation for why there is more vasospasm after subarachnoid hemorrhage in individuals and transgenic mice homozygous for the Hp 2 allele.     

Histochemical evidence for inducible nitric oxide synthase in advanced but non-ruptured human atherosclerotic carotid arteries

In response to cytokine stimulation, the inducible isoform of the nitric oxide synthase (iNOS) produces large amounts of nitric oxide with potential consequences in the pathophysiology of atherosclerosis. Previous investigations have demonstrated the presence of iNOS in human atherosclerotic lesions. The goal of this study was to evaluate the occurrence of the expression of iNOS in ruptured versus non-ruptured human carotid atherosclerotic plaques. Using plastic-embedded sections, we performed in situ hybridization and immunohistochemistry on very advanced atherosclerotic lesions type V (non-ruptured) and type VI (ruptured) from 12 atheromatous carotid arteries from endarterectomy and six non-atherosclerotic internal mammary arteries from aorto-coronary bypass. Only one internal mammary artery expressed iNOS in the endothelium. In contrast, iNOS mRNA and protein were repeatedly expressed in advanced lesions type V in 5/7 cases, particularly in inflammatory regions. Specific cell markers identified iNOS-positive cells as macrophages and T-lymphocytes but also as smooth muscle cells and endothelial cells adjacent to these inflammatory regions. Nitration of protein tyrosines was not always associated to iNOS expression but more likely to the presence of inflammatory cells. In complicated lesions type VI, the occurrence of iNOS mRNA and protein expression diminished drastically (1/5 cases). Combined expression of iNOS mRNA and protein is frequently found in advanced but non-ruptured human atherosclerotic carotid lesions while it becomes rare after the plaque has ruptured. These findings suggest that iNOS could be an active participant in the plaque rupture event.     

Hyaluronan fragments activate nitric oxide synthase and the production of nitric oxide by articular chondrocytes

Chondrocyte CD44 receptors anchor hyaluronan to the cell surface, enabling the assembly and retention of proteoglycan aggregates in the pericellular matrix. Hyaluronan-CD44 interactions also provide signaling important for maintaining cartilage homeostasis. Disruption of chondrocyte-hyaluronan contact alters CD44 occupancy, initiating alternative signaling cascades. Treatment with hyaluronan oligosaccharides is one approach to uncouple CD44 receptors from its native ligand, hyaluronan. In bovine articular chondrocytes, treatment with hyaluronan oligosaccharides or purified hyaluronan hexasaccharides induced the production of nitric oxide that mirrored nitric oxide production following interleukin-1 treatment. In contrast, 120 and 1,260 kDa hyaluronan did not induce production of nitric oxide. Human chondrocytes responded similarly to treatment with hyaluronan or hyaluronan oligosaccharides. Nitric oxide production from chondrocytes was mediated by activation of the inducible nitric oxide synthase, as confirmed by mRNA expression and inhibition of nitric oxide production by diphenyleneiodonium. Co-treatment of chondrocytes with hyaluronan oligosaccharides and interleukin-1 did not demonstrate additive effects. Blocking interleukin-1 receptors with an antagonist did not abolish the production of nitric oxide induced by treatment with hyaluronan oligosaccharides. Moreover, only COS-7 following transfection with a pCD44, not the CD44-null parental cells, responded to treatment with hyaluronan oligosaccharides by releasing nitric oxide. This study demonstrates a novel signaling potential by hyaluronan fragments, in lieu of endogenous hyaluronan-chondrocyte interactions, resulting in the activation of inducible nitric oxide synthase.