nitric oxide synthase (nos): oxygen...
TRANSCRIPT
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Nitric oxide (NO)
1. Nitric oxide synthase: Oxygen activation
2. NO2-, NO3-
3. NO-related reactions and functions
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Nitric Oxide (NO)
• An important signalling and cytotoxic molecule in the cardiovascular, nervous, and immune systems.
• From diabetes to hypertension, cancer to drug addiction, stroke to intestinal motility, memory and learning disorders to septic shock, sunburn to anorexia, male function to tuberculosis.
• Louis J. Ignarro, Robert F. Furchgott, and Ferid Murad have been jointly awarded the 1998 Nobel Prize in Physiology or Medicine for their discoveries concerning "nitric oxide as a signaling molecule in the cardiovascular system."
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The Nobel Prize in Physiology or Medicine 1998
The Nobel Assembly at the Karolinska Institute in Stockholm, Sweden,
has awarded the Nobel Prize in Physiology or Medicine for 1998 to Robert
F Furchgott, Louis J Ignarro and Ferid Murad for their discoveries
concerning "the nitric oxide as a signalling molecule in the cardiovascular
system".
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Conditions with Disruption of Vascular
NO as a Component
• Atherosclerosis including coronary, peripheral and cerebral artery diseases
• Hypercholesterolemia
• Hypertension
• Hyperhomocysteinemia
• Pulmonary hypertension
• Heart Failure
• Erectile Dysfunction
• Diabetes
• Raynaud’s disease
• Sepsis
• Sickel cell anemia
• Duchenne’s Muscular Dystrophy
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Figure 12. Summarized illustration of the main, significant categories of NO reactions in the vascular system. The reactions include (1) reactions with metal centers (mainly heme); (2) S-nitrosylation, or the interaction of NO with cysteine sulfahydryls/thiol, where a nitrosyl group is added post-translationally; (3) nitration (protein tyrosine); (4) free-radical interactions; (5) reactions with plasma O2; and (6) synthesis of cGMP through the catalysis of sGC by NO, then leading to the activation of protein kinases and phosphodiesterases.
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NO is synthesized
1. by Nitric Oxide Synthase (NOS)
2. fom NO2- (and NO3-)
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Nitric oxide synthase (NOS): Oxygen activation
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Nitric Oxide Synthase (NOS)
Heme Fe(III)-S--Cys
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Cytochrome P450: Cysteine-S binding to Fe(II) heme is important for
activation of O2.
Cytochrome c, Cytochrome b5: Electron-transfer relating heme proteins.
Myoglobin and hemoglobin: Histidine-midazole binding to Fe(II) heme
is important for O2 storage and O2 carrier, respectively. 10
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R: substrate
R: substrate
R: substrate
R: substrate
R: substrate
Shunt reaction
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Catalysis of Nitric Oxide Synthase (NOS)
O O
P450: Fe(III)heme-Cys, Reductase NOS: Fe(III)heme-Cys, Calmodulin H4B, fused enzyme with reductase, dimer 13
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Role of Tetrahydrobiopterin (H4B)
Crane et al. Biochemistry, Vol.39, 4608-4621, 2000.
Possible Electron-Transfer during H4B Oxidation
HN
N NH
HN
O
NH2
OH
OH
HN
N NH
HN
O-
NH2
OH
OH
H
– e–
O
O
OH
O
+ e– hemeheme
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Role of Tetrahydrobiopterin (H4B)
Hurshman et al. Biochemistry, Vol. 38, 15689-15696,1999
H+
H2O
H3B •
H4B
FeIIIOOH
FeIIIO2
NHA
FeIIO2
FeIII
FeII
e–
O2
L-Arg
(FeO)3+
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Nitric Oxide Synthase (NOS)
• Neuronal NOS (nNOS): Ca2+, Calmodulin NO nM
• Endothelium NOS (eNOS): Ca2+, Calmodulin NO nM
• Inducible NOS (iNOS): Cytokines, endotoxin (LPS)
Transcription, much larger NO NO mM
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Figure 7. Nitric oxide synthase. (A) eNOS ranges from 135 to 160 kDa and has a dimeric structure. The dimerization is catalyzed by the binding of heme. Zn binding in the dimer interface help stabilize the dimer. This dimerization process was found to be crucial to the catalytic activity and H4B.H4B poses a permanently bound position to NOS as it cycles between a fully reduced and an electron-oxidized form. eNOS is the major form of NOS that is involved in the vascular system and is highly dependent on calcium, thus facilitating calcium-concentration-specific modest synthesis of NO. (B) NO is generated through oxidation of the terminal guanidino nitrogen of l-arginine (l-arg) in the presence of dimerized NOS with the association of cofactors, including nicotinamide adenine dinucleotide phosphate hydrogen, flavin adenine dinucleotide, and flavin mononucleotide. In addition to the essential cofactors, chemical stimuli such as acetylcholine, bradykinin, estrogensphingosine 1-phosphate, H2O2, and angiotensin II and mechanical stimuli such as laminar shear stress and cyclic strain can also influence eNOS activation. (C) NO synthesis catalyzing cofactor, tetrahydrobiopterin H4B.
Chem. Rev. 111, 5742 (2011) Nitric oxide: A guardian for vascular grafts?
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Schematic Diagram of NOS and Hypothetical Electron
Transfers
Heme
Oxygenase
domain
Reductase
domain
e-
Heme
e-
CaM
CaM
CaM binding
domain
nNOS:
160 kDa subdomaim
Homodimer
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H4B and polar amino acids are important for
electron transfer reaction
Interface structure of the heme binding domains
of the two subunits 20
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Caveolae Outer leaflet
Cytoplasmic leaflet
Caveolin-1 nNOS
inactive CaM
Ca2+
inactive
CaM
active
active
CaM
nNOS ·NO L-Citrulline
L-Arginine 1.5 NADPH
1.5 NADP+
inactive
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NO from NO2- and NO3-
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28 Nature Chemical Biology 5, 865 (2009) Meeting Report
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Ion implicated in blood pact Nature Medicine 9, 1460
(2003)
The emerging biology of the nirite anion NO2-
Nature Chemical Biology 1, 308 (2005) 29
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Nature Chemical Biology 3, 785 (2007)
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Nature Review Neuroscience 8, 766 (2007).
NO in the central nervous system: neuroprotection versus
neurotoxicity.
Nature Chemical Biology 3, 678, (2007).
Nitrated cyclic GMP as a new cellular signal.
Nature Review Drug Discovery 7, 156 (2008).
The nitrate-nitrite-nitric oxide pathway in physiology and
therapeutics
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Dual Role of Nitric Oxide Production in
Cerebral Ischemic Injury
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No more toxic SNO: signal transduction
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Chemical Reactivity in the Chemical
Biology of Nitric Oxide
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Probably, minor
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The Indirect Effects of Nitric Oxide
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The Effects of Nitrosactive Stress
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NO carrier NO-glutathione Probably, no N2O3
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The Oxidative Chemistry of Nitroxyl
Proc. Nat. Acad. Sci. USA 101, 4003 (2004)
Tyr nitration by NO2. 37
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Figure 12. Summarized illustration of the main, significant categories of NO reactions in the vascular system. The reactions include (1) reactions with metal centers (mainly heme); (2) S-nitrosylation, or the interaction of NO with cysteine sulfahydryls/thiol, where a nitrosyl group is added post-translationally; (3) nitration (protein tyrosine); (4) free-radical interactions; (5) reactions with plasma O2; and (6) synthesis of cGMP through the catalysis of sGC by NO, then leading to the activation of protein kinases and phosphodiesterases.
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Major functions of NO are
-SNO formation
Activation of soluble guanylate cyclase (sGC)
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Important roles of
-Cys-S-NO: SNO: Snow
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Figure 9. Red blood cell interactions with NO include (1) NO binding to the deoxygenated heme in oxygenated red blood cells, forming iron-nitrosyl hemoglobin (FeII–NO); (2) oxy-hemoglobin scavenging NO and transferring it to the β-globin Cys-93 residue to form SNOHb (NO transport); (3) hemoglobin deoxygenation and structural transitions from R (oxy) to T (deoxy) facilitating the release of NO; and (4) T-state Hb reacting with NO species and undergoing Hb nitrosylation. Note: In the R state, Cys β93 is enclosed in a hydrophobic pocket, and the heme pocket is more accessible. In the T state, Cys β93 is exposed to reactions, and the heme pocket is less accessible.
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Mol. Cell 43, 1 (2011)
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J. Biol. Chem. 287, 4411 (2012). Regulation by S-Nitrosylation of Protein Post-translational Modification Protein post-translational modification by S-nitrosylation conveys a ubiquitous influence of nitric oxide on signal transduction in eukaryotic cells. The wide functional purview of S-nitrosylation reflects in part the regulation by S-nitrosylation of the principal protein post-translational modifications that play a role in cell signaling, including phosphorylation, acetylation, ubiquitylation and related modifications, palmitoylation, and alternative Cys-based redox modifications. In this minireview, we discuss the mechanisms through which S-nitrosylation exerts its broad pleiotropic influence on protein post-translational modification.
Fig. 1 Schematic summary of principal post-translational mechanisms regulated by S-nitrosylation and molecular loci of regulation.
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S-nitrosylation: integrator of cardiovascular performance and oxygen delivery J. Clin. Invest. 123, 101 (2013)
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Current Opio. Chem. Biol. 16, 498 (2012)
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Figure 1. Potential mechanisms of protein nitrosothiol formation and degradation. Activation of nitric oxide synthases (NOS) results in NO production and auto-S-nitrosation. S-Nitrosoglutathione (GSNO) or protein nitrosothiols can be generated from NO through either a transition metal catalyzed pathway, thiyl radical recombination, NO oxidation to N2O3, or transnitrosation with protein nitrosothiols such as NOS. Once formed, GSNO or S-nitrosated NOS can transfer nitrosothiols to other proteins through transnitrosation reactions. Degradation of nitrosothiols can occur through direct denitrosation of GSNO by the NAD(P)H-dependent enzymes GSNO reductase (GSNOR) or carbonyl reductase 1 (CBR1). Thioredoxin (Trx) can denitrosate protein nitrosothiols, a process that becomes catalytic through the activity of thioredoxin reductase (TrxR).
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Figure 2. Mechanisms of reactions discussed in this review. In the NO oxidation pathway NO first reacts with O2 to form a peroxynitrite radical. The peroxynitrite radical then undergoes radical recombination with NO to form ONOONO (or other isomers) followed by homolytic cleavage to form two molecules of NO2 radical. Radical recombination of NO and NO2 radicals results in N2O3 formation, which then reacts with a thiolate (RS
−) to form a nitrosothiol (RSNO) and nitrite (NO2
–). In the thiyl radical recombination pathway a thiyl radical is formed by hydrogen abstraction by another radical. Radical recombination of the thiyl radical with NO forms a nitrosothiol. Several transition metal catalyzed pathways are possible. In one pathway GSH weakly associates with cytochrome c, and the bound GSH attacks NO to form a GSNOH radical. The ferric heme of cytochrome c then accepts an electron from the GSNOH radical to form ferrous heme and GSNO. Reoxidation of cytochrome c completes the catalytic cycle. In another transition metal catalyzed pathway NO can bind to ferric heme, and the resulting Fe3+–NO complex (which possesses significant Fe2+-NO+ character) then reacts with a thiolate to form a nitrosothiol and ferrous heme. Once formed a nitrosothiol can be transferred through transnitrosation reactions in which a thiolate attacks the nitrogen atom of a nitrosothiol resulting in a nitroxyl disulfide intermediate or transition state that decays to a thiolate and a nitrosothiol. Denitrosation of nitrosothiols can proceed through several mechanisms. GSNOR and CBR1 catalyze hydride transfer from NAD(P)H to GSNO to form a GSNHOH intermediate that spontaneously rearranges to GSONH2. Thioredoxin catalyzed denitrosation proceeds through transnitrosation of a thioredoxin active-site cysteine (C32). Subsequently, the other active-site cysteine (C35) eliminates the nitrosothiol intermediate at C32 to form active-site oxidized thioredoxin and nitroxyl (NO−). The catalytic cycle is completed via thioredoxin reductase catalyzed NADPH-dependent reduction of the thioredoxin active-site disulfide.
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S-nitrosation of proteins relevant to Alzheimer’s disease during early stages of neurodegeneration Proc. Nat. Acad. Sci. USA 113, 4152 (2016) Significance Protein S-nitrosation (SNO-protein) is a posttranslational modification in which a cysteine (Cys) residue is modified by nitric oxide (SNO-Cys). SNO-proteins impact many biological systems, but their identification has been technically challenging. We developed a chemical proteomic strategy—SNOTRAP (SNO trapping by triaryl phosphine)—that allows improved identification of SNO-proteins by mass spectrometry. We found that S-nitrosation is elevated during early stages of neurodegeneration, preceding cognitive decline. We identified changes in the SNO-proteome during early neurodegeneration that are potentially relevant for synapse function, metabolism, and Alzheimer’s disease pathology. SNO-proteome analysis further reveals a potential linear motif for SNO-Cys sites that are altered during neurodegeneration. Our strategy can be applied to multiple cellular and disease contexts and can reveal signaling networks that aid drug development.
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Pathways affected by S-nitrosation during neurodegeneration.
Uthpala Seneviratne et al. PNAS 2016;113:4152-4157
©2016 by National Academy of Sciences
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SnapShot: Reactive
Oxygen Intermediates
Cell 140, March 19, 2010
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Poly(acrylonitrile)(PAN)
> 80 days
> 200 days
J. Am. Chem. Soc. 129, 3786 (2007) NO-releasing Fabrics
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• Figure 1 Hematoxylin- and eosin-stained cross sections of rat carotid arteries 14 days after injury with a balloon catheter without (A) and with (B) simultaneous periadventitial application of PAN/NO powder. Arrows point to neointimal hyperplasia seen in the untreated animals.
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Endogenous S-nitrosothiols protect against myocardial injury
PNAS 106, 6297 (2009)
60 -/- mouse smaller infarctions 心筋梗塞
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Heme-assisted S-nitrosation of a proximal thiolate in a nitric oxide transport protein
Proc. Natl. Acad. Sci. USA 102, 594 (2005)
61 Rhodnius prolixus (the kissing bug)
Cimex lectularius (the bedbug)
NO: Anti leukocyte adhension, Platelet Inhibition
Vasodilation, Smooth muscle relaxation, Blood
pressure
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Figure 12. Summarized illustration of the main, significant categories of NO reactions in the vascular system. The reactions include (1) reactions with metal centers (mainly heme); (2) S-nitrosylation, or the interaction of NO with cysteine sulfahydryls/thiol, where a nitrosyl group is added post-translationally; (3) nitration (protein tyrosine); (4) free-radical interactions; (5) reactions with plasma O2; and (6) synthesis of cGMP through the catalysis of sGC by NO, then leading to the activation of protein kinases and phosphodiesterases.
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Science 322, 1337 & 1392 (2008)
Same chemical strategy. Under aerobic conditions, host cells produce nitric oxide (NO) via the enzyme inducible nitric oxide synthase (iNOS). NO can kill replicating bacteria. Under hypoxic conditions, bacteria enter a state of nonreplicative persistence and host cell NO production is limited. Certain nitroimidazoles can generate NO (and other reactive nitrogen intermediates) within the bacteria under both aerobic and hypoxic conditions. Under hypoxic
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Nitrate and nitrite in biology, nutrition and therapeutics
Nature Chemical Biology 5, 865 (2009) Meeting Report
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Pathophysiologic model of
hyperoxia-mediated increase in
cardiomyocyte survival after
ischemia. Transient Hyperoxic Reoxygenation Reduces Cytochrome c Oxidase Activity by Increasing Superoxide Dismutase and Nitric Oxide J. Biol. Chem. 285, 11172 (2010)
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A SNO Storm in Skeletal Muscle
Cell 133, 33 (2008)
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Hypernitrosylated ryanodine receptor calcium release channels are leaky in dystrophic muscle Nature Medicine 15, 325 (2009)