Methylation of nitro sugars with diazomethane,and preparation of some new amino sugar methyl ethers

Diazomethane reacted with methyl 3,6-dideoxy-3-nitro-α-l-glucopyranoside (1) under catalysis by boron trifluoride to give the 2-O-methyl and the 2,4-di-O-methyl derivative (2 and 3). Similarly, the 4-acetate (4) of 1 afforded the 4-acetate (5) of 2. Boron trifluoride-catalyzed acetylation of 2 at about ?60° gave 5 whereas, at 0°, acetolysis took place producing 1,4-di-O-acetyl-3,6-dideoxy-2-O-methyl-3-nitro-α-l-glucopyranose (6). Diazomethane treatment of methyl 3,4,6-trideoxy-3-nitro-α-l-erythro- and -α-l-threo-hex-3-enopyranosides 7 and 8 furnished the corresponding 2-O-methyl derivatives 9 and 10. With triphenylphosphine and carbon tetrachloride, 2 yielded methyl 4-chloro-3,4,6-trideoxy-2-O-methyl-3-nitro-α-l-galactopyranoside (11) which was dehydrochlorinated to 9. Borohydride reduction of 9 gave methyl 3,4,6-trideoxy-2-O-methyl-3-nitro-α-l-xylo-hexopyranoside (12). Catalytic hydrogenation of 3 and 12 afforded the corresponding amino sugar hydrochlorides 13 and 15. Treatment of 5 with ammonia gave a 4-amino-3-nitro glycoside (isolated as the hydrochloride 17) hydrogenation of which led to methyl 3,4-diamino-3,4,6-trideoxy-2-O-methyl-α-l-glucopyranoside dihydrochloride (19). The N-acetyl derivatives (14, 16, 18, and 20) of the four new amino sugars were prepared.     

Reaction of derivatives of methyl 2,3-O-benzylidene-6-deoxy-α-L-mannopyranoside with butyllithium: Synthesis of methyl 2, 6-dideoxy-4-O-methyl-α-L-erytho-hexopyranosid-3-ulose

Methyl 2,3-O-benzylidene-6-deoxy-α-L-mannopyranoside (2) reacted with butyllithium to give a mixture of 1,5-anhydro-3-C-butyl-1,2,6-trideoxy-L-ribo-hex-1-enitol (3) and its L-arabino analogue (4), together with methyl 2,3,6-trideoxy-α-L-erythro-hex-2-enopyranoside (5). In contrast, the 4-O-methyl analogue (8) of 2 was converted by butyllithium into methyl 2,6-dideoxy-4-O-methyl-α-L-erythro-hexo-pyranosid-3-ulose (9), which was further characterized as its oxime 10. The 4-O-benzyl analogue of 8, obtained as two separate diastereoisomers (6 and 7) differing in configuration at C-2 of the dioxolane ring, gave a complex mixture of products on treatment with butyllithium.     

Design,synthesis, neuroprotective,antibacterial activities and docking studies of novel thieno[2,3-d]pyrimidine-alkyne Mannich base and oxadiazole hybrids

A series of thieno[2,3-d]pyrimidine alkyne Mannich base derivatives (7a-e, 8a-e) and thieno[2,3-d]pyrimidine 1,3,4-oxadiazole derivatives (9a-e, 10a-e) have been synthesized and evaluated for their neuroprotective and neurotoxicity activities where 9a, 10d displayed good neuroprotection 10.6 and 11.88?µg/mL respectively against the H₂O₂ induced cell death at the EC₅₀ values and 9b, 9d showed respective toxic effects on PC12 cells at CC₅₀ 86.12 and 94.16?µg/mL. Compounds 9a, 9e, 10a and 10b showed strong antibacterial activity against two gram positive (S. aureus, B. subtilis) and two gram-negative strains (E. coli, P. aeruginosa) and showed good binding affinities with C(30) carotenoid dehydrosqualene synthase, Gyrase A and LpxC. This is the first report for the demonstration of thieno[2,3-d] pyrimidine derivatives as promising neuroprotective agents against H₂O₂ induced neurotoxicity on PC12 cells.     

A side-reaction in the methylation of galacturonate derivatives with diazomethane-boron trifluoride etherate

Partial p-nitrobenzoylation of methyl (methyl 2-O-methyl-α-d-galactopyranosid)uronate (1) gave the 3-p-nitrobenzoate 2 in good yield. Treatment of 2 or methyl (methyl 2,3-di-O-benzoyl-α-d-galactopyranosid)uronate (11) with diazomethane-BF₃-etherate gave, in addition to the expected 4-methyl ethers, by-products resulting from lengthening of the carbon chain. The by-products were formulated as derivatives of methyl 4,7-anhydro-α-d-galacto-heptopyranosid-6-ulose dimethy acetal on the basis of p.m.r. and i.r. spectral data, by analysis of their mass-spectral fragmentation pattern, and by chemical transformations.     

Study on the reaction mechanism and the static injection chemiluminescence method for detection of acetaminophen

Acetaminophen, also called paracetamol, is found in Tylenol, Excedrin and other products as over–the‐counter medicines. In this study, acetaminophen as a luminol signal enhancer was used in the chemiluminescence (CL) substrate solution of horseradish peroxidase (HRP) for the first time. The use of acetaminophen in the luminol–HRP–H₂O₂ system affected not only the intensity of the obtained signal, but also its kinetics. It was shown that acetaminophen was to be a potent enhancer of the luminol–HRP–H₂O₂ system. A putative enhancement mechanism for the luminol–H₂O₂–HRP–acetaminophen system is presented. The resonance of the nucleophilic amide group and the benzene ring of acetaminophen structure have a great effect on O‐H bond dissociation energy of the phenol group and therefore on phenoxyl radical stabilization. These radicals act as mediators between HRP and luminol in an electron transfer reaction that generates luminol radicals and subsequently light emission, in which the intensity of CL is enhanced in the presence of acetaminophen. In addition, a simple method was developed to detect acetaminophen by static injection CL based on the enhanced CL system of luminol–H₂O₂–HRP by acetaminophen. Experimental conditions, such as pH and concentrations of substrates, have been examined and optimized. The proposed method exhibited good performance, the linear range was from 0.30 to 7.5 mM, the relative standard deviation was 1.86% (n = 10), limit of detection was 0.16 mM and recovery was 99 ± 4%. Copyright © 2013 John Wiley & Sons, Ltd.     

Reactivation of horseradish peroxidase with imidazole for continuous determination of hydrogen peroxide using a microflow injection–chemiluminescence detection system

A method for reactivation of inactivated horseradish peroxidase (HRP) was studied and exploited in an assay for hydrogen peroxide (H₂O₂). Addition of imidazole into a mobile phase made continuous determination of hydrogen peroxide (H₂O₂) possible by micro?ow injection based on horseradish‐catalysed luminol chemiluminescence. For reproducible determination of H₂O₂ with HRP, the inactivation of HRP via protonation of the active sites of HRP caused by reaction with H₂O₂ must be avoided. We successfully reactivated protonated HRP (inactive HRP) with exogenous imidazole in the mobile phase of the micro?ow injection system. The imidazole successfully removed the attached proton from the inactive sites of the HRP. This assay was reproducible (within‐run reproducibility, CV = 4.0%) and the detection limit for H₂O₂ was 5 pmol. Copyright © 2003 John Wiley & Sons, Ltd.     

Nucleophilic displacements of methyl 2,3-O-isopropylidene-4-O-toluene-p-sulphonyl-α-d-lyxo- and -β-l-ribo-pyranosides,and deamination of the corresponding 4-amino sugars

Reinvestigation of the reaction of methyl 2,3-O-isopropylidene-4-O-toluene-p-sulphonyl-α-d-lyxopyranoside (4) with azide ion has shown that methyl 4-deoxy-2,3-O-isopropylidene-β-l-erythro-pent-4-enopyranoside (8, ～51.5%) is formed, as well as the azido sugar 7 (～48.5%) of an S_N2 displacement. The unsaturated sugar 8 was more conveniently prepared by heating the sulphonate 4 with 1,5-diazabicyclo-[5.4.0]undec-5-ene. An azide displacement on methyl 2,3-O-isopropylidene-4-O-toluene-p-sulphonyl-β-l-ribopyranoside (12) furnished methyl 4-azido-4-deoxy-2,3-O-isopropylidene-α-d-lyxopyranoside (13, ～66%) and the unsaturated sugar 14 (～28.5%), which was also prepared by heating the sulphonate with 1,5-diazabicyclo[5.4.0]undec-5-ene. Deamination of methyl 4-amino-4-deoxy-2,3-O-isopropylidene-α-d-lyxopyranoside (5), prepared by reduction of 13, with sodium nitrite in 90% acetic acid at ～0°, yielded methyl 2,3-O-isopropylidene-α-d-lyxopyranoside (10a, 26.2%), methyl 2,3-O-isopropylidene-β-l-ribofuranoside (21a, 18.4%), and the corresponding acetates 10b (34.5%) and 21b (21.3%). These products are considered to arise by solvolysis of the bicyclic oxonium ion 29, formed as a consequence of participation by the ring-oxygen atom in the deamination reaction. Similar deamination of methyl 4-amino-4-deoxy-2,3-O-isopropylidene-β-l-ribopyranoside (6) afforded, exclusively, the products 10a (34.4%) and 10b (65.6%) of inverted configuration. Deamination of methyl 5-amino-5-deoxy-2,3-O-isopropylidene-β-d-ribofuranoside (20) gave 22ab, but no other products. An alternative synthesis of the amino sugars 5 and 6 is available by conversion of 10a into methyl 2,3-O-isopropylidene-β-l-erythro-pentopyranosid-4-ulose (11), followed by reduction of the derived oxime 15 with lithium aluminium hydride.     

The deamination of methyl 5-amino-5,6-dideoxy-2,3-O-isopropylidene-α-L-talo- and -β-D-allo-furanoside with nitrous acid

Deamination of methyl 5-amino-5,6-dideoxy-2,3-O-isopropylidene-α-L-talofuranoside (6) with sodium nitrite in 90% acetic acid at ≈0° gave methyl 6-deoxy-2,3-O-isopropylidene-α-L-talofuranoside (8a) and methyl 6-deoxy-2,3-O-isopropylidene-β-D-allofuranoside (9a) (combined yield, 12.3%), the corresponding 5-acetates 8b (2.9%) and 9b (26.4%), and the unsaturated sugar methyl 5,6-dideoxy-2,3-O-isopropylidene-β-D-ribo-hex-5-enofuranoside (10) (43.5%). Similar deamination of methyl 5-amino-5,6-dideoxy-2,3-O-isopropylidene-β-D-allofuranoside (7) gave 8a and 9a (combined yield, 20.4%), 8b (12.5%), 9b (25.8%), 10 (7.7%), and the rearranged products 6-deoxy-2,3-O-isopropylidene-5-O-methyl-L-talofuranose (13a, 17.5%) and the corresponding 1-acetate 13b (14.1%). A synthesis of 13a was accomplished by successive methylation and debenzylation of the conveniently prepared benzyl 6-deoxy-2,3-O-isopropylidene-α-L-talofuranoside (15b). Differences between the two sets of deamination products can be rationalized by assuming that the carbonium-ion intermediate reacts in the initial conformation assumed, before significant interconversion to other conformations occurs.     

Hydrogen peroxide and nitric oxide promote reproductive growth in Litchi chinensis

Vegetative growth and reproductive growth strongly competes with each other during panicle development in litchi (Litchi chinensis Sonn.). We herein investigated the roles of hydrogen peroxide and nitric oxide in the competition between growth of rudimentary leaves and panicle development. The results show that the chilling-induced flowering increased H₂O₂ and NO contents in the mixed buds. Treatments with sodium nitroprusside (SNP), the NO donor, and methyl viologen dichloride hydrate (MV), the superoxide generator, increased NO and H₂O₂ contents in the mixed buds. MV and SNP treatments promoted abscission of rudimentary leaves and encouraged panicle development before or at the stage of panicle emergence. The nitric oxide synthase inhibitor N ^ω -nitro-L-arginine methyl ester (L-NAME) and the H₂O₂ trapper dimethylthiourea (DMTU) inhibited a chilling-induced flowering. SNP promoted the expression of litchi LEAFY homolog (LcLFY). These promotive effects were suppressed by the NO scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl3-oxide (PTIO) and the H₂O₂ trapper, DMTU. The results suggest that H₂O₂ and NO promote reproductive growth by inhibiting the growth of rudimentary leaves as well as by promoting the expression of the flower related gene, LcLFY.     

Synthesis and reactions of α- and β-d-glucopyranosyluronic esters of amino acids

The synthesis of the fully benzylated α- and β-d-glucopyranosyluronic esters of 1-benzyl N-benzyloxycarbonyl-l-aspartic and -glutamic acids and N-(tert-butoxycarbonyl)-l-phenylalanine, followed by hydrogenolysis, afforded the respective anomers of the 1-O-acyl-d-glucopyranuronic acids 2, 7, and 12. Esterification of both anomers of the N-acetylated derivatives of 2 and 7 by diazomethane was accompanied by glycosyl-bond cleavage, and, in the case of the α anomers, with concomitant 1→2 acyl migration to give, after O-acetylation, the 2-O-acyl O-acetyl methyl ester derivatives 5 and 10, respectively. Similarly, 12α yielded methyl 1,3,4-tri-O-acetyl-2-O-[N-(tert-butoxycarbonyl)-l-phenylalanyl]-d-glucopyranuronate and an analogue having a furanurono-6,3-lactone structure. Esterification of the C-5 carboxyl group, in 1-O-acyl-α-d-glucopyranuronic acids by methanol in the presence of the BF_3^?-MeOH reagent (1–1.5 equiv.) proceeded without acyl migration. By using this procedure, followed by acetylation, the N-acetylated derivative of 7α afforded methyl 2,3,4-tri-O-acetyl-1-O-(1-methyl N-acetyl-l-glutam-5-oyl)-α-d-glucopyranuronate, and 12α gave methyl 2,3,4-tri-O-acetyl-1-O-(N-acetyl-l-phenylalanyl)-α-d-glucopyranuronate; the formation of the latter involved cleavage of the tert-butoxycarbonyl group by BF₃, followed by N-acetylation in the next step.     

Inertness of horseradish peroxidase to 2,4-dichlorophenoxyacetic acid

The possibility of mutual effects of 2,4-D and horseradish (Armoracia lapathifolia L.) peroxidase on each other has been explored by four procedures. (i) Compounds I, II, and III of horseradish peroxidase (HRP) and H₂O₂ were exposed to 2,4-D. (ii) Extracts from batchwise operations of HRP + H₂O₂ and 2,4-D were analyzed for oxidation products by means of thin layer chromatography. (iii) The velocity of the IAA oxidase reaction with HRP as catalyst, and (iv) K_m and V_s of the overall peroxidation of guaiacol by HRP + H₂O₂, were determined in the absence and presence of 2,4-D. The results failed to show any effect of 2,4-D; only at very high concentrations did 2,4-D slightly inhibit the oxidation of IAA by one isoperoxidase. It is concluded that 2,4-D does not promote growth in plants by hampering a peroxidase-catalyzed IAA oxidation. It seems probable that 2,4-D perturbs the isoperoxidase pattern by acting at some step prior to the release of the enzyme from its site of synthesis.     

Mono- and bis-cobalt complexes with carbonyl, nitrosyl, and monocarbollide ligands

Reaction of [Co(CO)₃(NO)] with [2-NMe₃-closo-2-CB₁₀H₁₀] in refluxing CH₂Cl₂ affords the mono- and di-cobalt complexes [1-NMe₃-2-CO-2-NO-closo-2,1-CoCB₁₀H₁₀] (3) and [2,7-{Co(CO)(NO)}-7-(μ-H)-1-NMe₃-2-CO-2-NO-closo-2,1-CoCB₁₀H₉] (4), respectively, of which 4 contains formally both Co(I) and Co(-I) centers. Compound 4 reacts with CO to give 3, or with donor ligands L in the presence of Me₃NO to afford simple substituted species, [1-NMe₃-2-L-2-NO-closo-2,1-CoCB₁₀H₁₀] (compounds 5; L = PEt₃, PPh₃, CNBu^t).     

Intragastric nitration by dietary nitrite: implications for modulation of protein and lipid signaling

Inorganic nitrite, derived from the reduction of nitrate in saliva, has recently emerged as a protagonist in nitric oxide (^?NO) biology as it can be univalently reduced to ^?NO, in the healthy human stomach. Important physiological implications have been attributed to nitrite-derived ^?NO in the gastrointestinal tract, namely modulation of host defense, blood flow, mucus formation and motility. At acidic pH, nitrite generates different nitrogen oxides depending on the local microenvironment (redox status, gastric content, pH, inflammatory conditions), including ^?NO, nitrogen dioxide (^?NO₂), dinitrogen trioxide (N₂O₃), and peroxynitrite. Thus, the gastric environment is a significant source of nitrating and nitrosating agents, especially in individuals consuming a nitrate/nitrite-rich diet on a daily basis. Both, the gastric lumen and mucosa contain putative targets for nitration, not only proteins and lipids from ingested aliments but also endogenous proteins secreted by the oxyntic glands. The physiological and functional consequences of nitration of gastric mediators will impact on local processes including food digestion and ulcerogenesis. Additionally, gastric nitration products (such as nitrated lipids) may be absorbed and affect systemic pathways. Thus, dietary ingestion of nitrate will have direct consequences for endogenous protein nitration, as indicated by our preliminary data.     

Enhanced detection of H2O2 in cells expressing Horseradish Peroxidase

A new procedure for fluorescent detection of intracellular H₂O₂ in cells transiently expressing the catalyst Horseradish Peroxidase (HRP) is setup and validated. More specific reaction with HRP largely amplifies oxidation of the redox probes used (2′,7′-dichlorodihydrofluorescein and dihydrorhodamine). Expression of HRP does not affect cell viability. The procedure reveals MAO activity, a primary intracellular H₂O₂ source, in monolayers of intact transfected cells. The probes oxidation rate responds specifically to the MAO activation/inhibition. Their oxidation by MAO-derived H₂O₂ is sensitive to intracellular H₂O₂ competitors: it decreases when H₂O₂ is removed by pyruvate and it increases when the GSH-dependent removal systems are impaired. Specific response was also measured after addition of extracellular H₂O₂. Oxidation of the fluorescent probes following reaction of H₂O₂ with endogenous HRP overcomes most criticisms in their use for intracellular H₂O₂ detection. The method can be applied for direct determination in plate reader and is proposed to detect H₂O₂ generation in physio-pathological cell models.     

Signal interaction between nitric oxide and hydrogen peroxide in heat shock-induced hypericin production of <Emphasis Type="Italic">Hypericum perforatum suspension</Emphasis> cells

Heat shock (HS, 40°C, 10 min) induces hypericin production, nitric oxide (NO) generation, and hydrogen peroxide (H₂O₂) accumulation of Hypericum perforatum suspension cells. Catalase (CAT) and NO specific scavenger 2–4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) suppress not only the HS-induced H₂O₂ generation and NO burst, but also the HS-triggered hypericin production. Hypericin contents of the cells treated with both NO and H₂O₂ are significantly higher than those of the cells treated with NO alone, although H₂O₂ per se has no effects on hypericin production of the cells, which suggests the synergistic action between H₂O₂ and NO on hypericin production. NO treatment enhances H₂O₂ levels of H. perforatum cells, while external application of H₂O₂ induces NO generation of cells. Thus, the results reveal a mutually amplifying action between H₂O₂ and NO in H. perforatum cells. CAT treatment inhibits both HS-induced H₂O₂ accumulation and NO generation, while cPTIO can also suppress H₂O₂ levels of the heat shocked cells. The results imply that H₂O₂ and NO may enhance each other’s levels by their mutually amplifying action in the heat shocked cells. Membrane NAD(P)H oxidase inhibitor diphenylene iodonium (DPI) and nitric oxide synthase (NOS) inhibitor S,S′-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea (PBITU) not only inhibit the mutually amplifying action between H₂O₂ and NO but also abolish the synergistic effects of H₂O₂ and NO on hypericin production, showing that the synergism of H₂O₂ and NO on secondary metabolite biosynthesis might be dependent on their mutual amplification. Taken together, data of the present work demonstrate that both H₂O₂ and NO are essential for HS-induced hypericin production of H. perforatum suspension cells. Furthermore, the results reveal a special interaction between the two signal molecules in mediating HS-triggered secondary metabolite biosynthesis of the cells.     

A Highly Sensitive and Selective Hydrogen Peroxide Biosensor Based on Gold Nanoparticles and Three-Dimensional Porous Carbonized Chicken Eggshell Membrane

A sensitive and noble amperometric horseradish peroxidase (HRP) biosensor is fabricated via the deposition of gold nanoparticles (AuNPs) onto a three-dimensional (3D) porous carbonized chicken eggshell membrane (CESM). Due to the synergistic effects of the unique porous carbon architecture and well-distributed AuNPs, the enzyme-modified electrode shows an excellent electrochemical redox behavior. Compared with bare glass carbon electrode (GCE), the cathodic peak current of the enzymatic electrode increases 12.6 times at a formal potential of −100mV (vs. SCE) and charge-transfer resistance decreases 62.8%. Additionally, the AuNPs-CESM electrode exhibits a good biocompatibility, which effectively retains its bioactivity with a surface coverage of HRP 6.39×10⁻⁹ mol cm⁻² (752 times higher than the theoretical monolayer coverage of HRP). Furthermore, the HRP-AuNPs-CESM-GCE electrode, as a biosensor for H₂O₂ detection, has a good accuracy and high sensitivity with the linear range of 0.01–2.7 mM H₂O₂ and the detection limit of 3μM H₂O₂ (S/N = 3).     

Preparation of Methyl 4,6-O-benzylidene-2-deoxy-2-nitro-β-D-glucopyranoside from the corresponding 3-deoxy-3-nitro derivatives with sodium nitrite

Treatment of methyl 4,6-O-benzylidene-2,3-dideoxy-3-nitro-β-D-erythro-hex-2-enopyranoside (2) with nitrous acid afforded the title 2-nitro sugar (4). The same product was also prepared by heterogeneous reaction of methyl 2-O-acetyl-4,6-O-benzylidene-3-deoxy-3-nitro-β-D-glucopyranoside (1) with sodium nitrite in the presence of a phase-transfer catalyst. Acid hydrolysis of 4 gave methyl 2-deoxy-2-nitro-β-D-glucopyranoside (7). Acetylation of 4, followed by elimination of acetic acid, afforded a 2-nitroalkene (6). 71e 3-acetate 5 reacted with ammonia, dimethylamine, and 2,4-pentanedione to give the products 8, 9, and 10, respectively, having the gluco configuration.     

Synthesis of monodeoxy and mono-O-methyl congeners of methyl β-d-mannopyranosyl-(1→2)-β-d-mannopyranoside for epitope mapping of anti-Candida albicans antibodies

A panel of six complementary monodeoxy and mono-O-methyl congeners of methyl β-d-mannopyranosyl-(1→2)-β-d-mannopyranoside (1) were synthesized by stereoselective glycosylation of monodeoxy and mono-O-methyl monosaccharide acceptors with a 2-O-acetyl-glucosyl trichloroacetimidate donor, followed by a two-step oxidation-reduction sequence at C-2′. The β-manno configuration of the final deprotected congeners 2-7 was confirmed by measurement of ¹J_C1,H1 heteronuclear and ³J_1′,2′ homonuclear coupling constants. These disaccharide derivatives will be used to map the epitope recognized by a protective anti-Candida albicans monoclonal antibody C3.1 (IgG3) and to determine its key polar contacts with the binding site.     

Reaction of nitric oxide with the oxidized di-heme and heme–copper oxygen-reducing centers of terminal oxidases: Different reaction pathways and end-products

Inhibition of terminal oxidases by nitric oxide (NO) has been extensively investigated as it plays a role in regulation of cellular respiration and pathophysiology. Cytochrome bd is a tri-heme (b₅₅₈, b₅₉₅, d) bacterial oxidase containing no copper that couples electron transfer from quinol to O₂ (to produce H₂O) with generation of a transmembrane protonmotive force. In this work, we investigated by stopped-flow absorption spectroscopy the reaction of NO with Escherichia coli cytochrome bd in the fully oxidized (O) state. We show that under anaerobic conditions, the O state of the enzyme binds NO at heme d with second-order rate constant k_on = 1.5 ± 0.2 × 10² M⁻¹ s⁻¹, yielding a nitrosyl adduct (d³⁺–NO or d²⁺–NO⁺) with characteristic optical features (an absorption increase at 639 nm and a red shift of the Soret band). The reaction mechanism is remarkably different from that of O cytochrome c oxidase in which the heme–copper binuclear center reacts with NO approximately three orders of magnitude faster, forming nitrite. The data allow us to conclude that in the reaction of NO with terminal oxidases in the O state, Cu_B is indispensable for rapid oxidation of NO into nitrite.