Presence Of H218O Research Articles

A Water-Promoted Mars-van Krevelen Reaction Dominates Low-Temperature CO Oxidation over Au-Fe2O3 but Not over Au-TiO2.

We provide experimental evidence that is inconsistent with often proposed Langmuir-Hinshelwood (LH) mechanistic hypotheses for water-promoted CO oxidation over Au-Fe2O3. Passing CO and H2O, but no O2, over Au-γ-Fe2O3 at 25 °C, we observe significant CO2 production, inconsistent with LH mechanistic hypotheses. Experiments with H218O further show that previous LH mechanistic proposals cannot account for water-promoted CO oxidation over Au-γ-Fe2O3. Guided by density functional theory, we instead postulate a water-promoted Mars-van Krevelen (w-MvK) reaction. Our proposed w-MvK mechanism is consistent both with observed CO2 production in the absence of O2 and with CO oxidation in the presence of H218O and 16O2. In contrast, for Au-TiO2, our data is consistent with previous LH mechanistic hypotheses.

High Selectivity to HCl for the Catalytic Removal of 1,2-Dichloroethane Over RuP/3DOM WOx: Insights into the Effects of P-Doping and H2O Introduction.

Ru-based catalysts for catalytic combustion of high-toxicity Cl-containing volatile organic compounds are inclined to produce Cl2 instead of ideal HCl due to the Deacon reaction. We herein reported that the three-dimensionally ordered macroporous (3DOM) WOx-supported RuP nanocatalyst greatly improved HCl selectivity (at 400 °C, increased from 66.0% over Ru/3DOM WOx to 96.4% over RuP/3DOM WOx) and reduced chlorine-containing byproducts for 1,2-dichloroethane (1,2-DCE) oxidation. P-doping enhanced the number of structural hydroxyl groups and Brønsted acid sites. The isotopic 1,2-DCE temperature-programmed desorption experiment in the presence of H218O indicated the generation of a new active oxygen species 16O18O that participated in the reaction. Generally, P-doping and H2O introduction could promote the exchange reaction between Cl and hydroxyl groups, rather than oxygen defects, and then benefit the production of HCl and reduce the generation of other chlorine species or Cl2, via the reaction processes of C2H3Cl → alcohol → aldehyde → carboxylic acids.

Highly Efficient Aromatic C−H Oxidation with H2O2 in the Presence of Iron Complexes of the PDP Family

AbstractThe catalytic activity of a series of iron complexes of the PDP family (PDP=N,N′‐bis(2‐pyridylmethyl)‐2,2′‐bipyrrolidine) in the oxidation of aromatic substrates with H2O2 has been studied. In the presence of acetic acid, these complexes efficiently catalyze the oxidation of benzene and alkylbenzenes with high selectivity for oxygen incorporation into the aromatic ring (up to 93 %), performing up to 84 catalytic turnovers. The parent complex, [(PDP)(OTf)2], has demonstrated the highest catalytic efficiency and aromatic oxidation selectivity. The yield of products of oxidation of different substrates increases in line with increasing number of electron‐donating alkyl groups of the substrates: halogenbenzenes<benzene<monoalkylbenzenes<dialkylbenzenes, which, together with the results of competitive oxidation experiments and the observation of inverse primary kH/kD (0.90–0.92), agrees with the SEAr oxidation mechanism. Low‐temperature EPR studies have witnessed the presence of low‐spin (g1=2.071, g2=2.008, g3=1.960) perferryl intermediates, demonstrating direct reactivity toward benzene. The oxidation of m‐xylene in the presence of H218O has shown the same probability of 18O incorporation into the aromatic ring and the aliphatic moieties, which is indicative that both the aromatic and aliphatic oxidations are conducted by the same active species.

Glycoproteins in the Midgut Microvilar Membrane of Spodoptera frugiperda (Lepidoptera: Noctuidae)

Spodoptera frugiperda, the fall armyworm, is an agricultural pest widely distributed from South to North America. The use of specific and environmentally friendly pesticides such as Cry toxins produced by the bacteria Bacillus thuringiensis is an important way to control these animals. There is evidence that these toxins bind to membrane‐bound proteins (named Cry toxin receptors‐CTR) of the midgut microvilar epithelium (MME), leading to cell lysis or activating cell death pathways. Among the recognition mechanisms, it has been reported that domain III of Cry1ac interacts with glycans attached to CTR, and that the presence of N‐acetylgalactosamine enhances pore‐formation activity. Nevertheless, only one published report describes glycan structures present for the CTR aminopeptidase N (APN). Moreover, there is still a huge lack of information about glycan composition in insects, which may differ in composition from mammalian ones. Thus, this study aims to do a broad characterization of glycoproteins membrane‐bounded to the midgut microvilar epithelium of S. frugiperda.Isolated MME was submitted to both in‐gel and in‐solution tryptic digestions. Gel band digests were separately submitted either to online HILIC/C18 LC‐MS/MS in an Agilent 6550 Q‐TOF/TOF instrument or to reversed phase LC‐MS/MS into a Q Exactive HF (Thermo). A fraction of each of the same gel band digests was treated with Glycosidase A and analyzed in the latter instrument. Permethylation of released glycans was done after in‐solution digestion and treatment with Glycosidase A in the presence of H218O. Further analysis of permethylated glycans was accomplished by MALDI‐MS in an UltrafleXtreme (Bruker). Spectra assignments were done either manually or using Peaks® 8 (BSI).A total of 16 different glycoproteins (21 glycopeptides) were identified in S. frugiperda MME, including two unknown proteins. Four APNs and one alkaline phosphatase, already known CTRs, are among the identified glycoproteins. In more than one case, an APN glycopeptide showed multiple glycoforms. For instance, peptide 99INVVNNTNGENVQLR113 was identified with Hex9HexNAc2, Hex3HexNAc2dHex5‐6, Hex4HexNAc3dHex2, among others. Transferrin was shown to be heavily glycosylated, displaying four different glycopeptides. The most commonly observed structure was the high mannose glycan (peptides 456LIQNQPINATR466 and 624ALCRNNSLALR634), but fucosylated glycans Hex3HexNAc2dHex2 and Hex4HexNAc2dHex2 were found in peptides 473ESSVVSGNVSR483 and 241VNANATNTAVAYVAWQHVR259, respectively. The assignments of most abundant glycan structures were confirmed after release and permethylation followed by MALDI‐MS analysis.Novel aspect: This is the first broad characterization of glycoproteins present in the midgut of a lepidopteran which will allow a better understand of possible Cry toxin targets and improve the knowledge about invertebrate glycans.Support or Funding InformationSupported by FAPESP (2014/14183‐2; 2016/09511‐6; 11/51685‐8) and NIH (P41 6M104603)

Combining recombinant ribonuclease U2 and protein phosphatase for RNA modification mapping by liquid chromatography–mass spectrometry

Ribonuclease (RNase) mapping of modified nucleosides onto RNA sequences is limited by RNase availability. A codon-optimized gene for RNase U2, a purine selective RNase with preference for adenosine, has been designed for overexpression using Escherichia coli as the host. Optimal expression conditions were identified enabling generation of milligram-scale quantities of active RNase U2. RNase U2 digestion products were found to terminate in both 2′,3′-cyclic phosphates and 3′-linear phosphates. To generate a homogeneous 3′-linear phosphate set of products, an enzymatic approach was investigated. Bacteriophage lambda protein phosphatase was identified as the optimal enzyme for hydrolyzing cyclic phosphates from RNase U2 products. The compatibility of this enzymatic approach with liquid chromatography–tandem mass spectrometry (LC–MS/MS) RNA modification mapping was then demonstrated. RNase U2 digestion followed by subsequent phosphatase treatment generated nearly 100% 3′-phosphate-containing products that could be characterized by LC–MS/MS. In addition, bacteriophage lambda protein phosphatase can be used to introduce 18O labels within the 3′-phosphate of digestion products when incubated in the presence of H218O, allowing prior isotope labeling methods for mass spectrometry to include digestion products from RNase U2.

Targeting Proteases in Cardiovascular Diseases by Mass Spectrometry-Based Proteomics

Proteases hydrolyze peptide bonds, thereby controlling the function of proteins and peptides on the posttranslational level. In the cardiovascular system, proteases play pivotal roles in the regulation of blood pressure, coagulation and other essential physiological processes. Accordingly, proteases are prime targets for therapeutic interventions and diagnostics. Proteases are part of complex proteolytic networks comprised of enzymes, inhibitors, activators, substrates and cleavage products. Analyzing these networks on a system-wide level is essential to understanding cardiovascular function and how dysregulation can lead to pathological conditions. Mass spectrometry-based quantitative and dynamic proteomics approaches are leading the way to enhance our knowledge of proteolytic networks such as the renin-angiotensin-system. Here, we critically review proteomics tools utilized in protease biology and provide an overview on how these methods can be used to characterize and validate protease function.

Mutagenesis of Surfactant Protein D Informed by Evolution and X-ray Crystallography Enhances Defenses against Influenza A Virus in Vivo

The recognition of influenza A virus (IAV) by surfactant protein D (SP-D) is mediated by interactions between the SP-D carbohydrate recognition domains (CRD) and glycans displayed on envelope glycoproteins. Although native human SP-D shows potent antiviral and aggregating activity, trimeric recombinant neck+CRDs (NCRDs) show little or no capacity to influence IAV infection. A mutant trimeric NCRD, D325A/R343V, showed marked hemagglutination inhibition and viral neutralization, with viral aggregation and aggregation-dependent viral uptake by neutrophils. D325A/R343V exhibited glucose-sensitive binding to Phil82 hemagglutinin trimer (HA) by surface plasmon resonance. By contrast, there was very low binding to the HA trimer from another virus (PR8) that lacks glycans on the HA head. Mass spectrometry demonstrated the presence of high mannose glycans on the Phil82 HA at positions known to contribute to IAV binding. Molecular modeling predicted an enhanced capacity for bridging interactions between HA glycans and D325A/R343V. Finally, the trimeric D325A/R343V NCRD decreased morbidity and increased viral clearance in a murine model of IAV infection using a reassortant A/WSN/33 virus with a more heavily glycosylated HA. The combined data support a model in which altered binding by a truncated mutant SP-D to IAV HA glycans facilitates viral aggregation, leading to significant viral neutralization in vitro and in vivo. These studies demonstrate the potential utility of homology modeling and protein structure analysis for engineering effective collectin antivirals as in vivo therapeutics.

2-Haloacrylate Hydratase, a New Class of Flavoenzyme That Catalyzes the Addition of Water to the Substrate for Dehalogenation

Enzymes catalyzing the conversion of organohalogen compounds are useful in the chemical industry and environmental technology. Here we report the occurrence of a new reduced flavin adenine dinucleotide (FAD) (FADH(2))-dependent enzyme that catalyzes the removal of a halogen atom from an unsaturated aliphatic organohalogen compound by the addition of a water molecule to the substrate. A soil bacterium, Pseudomonas sp. strain YL, inducibly produced a protein named Caa67(YL) when the cells were grown on 2-chloroacrylate (2-CAA). The caa67(YL) gene encoded a protein of 547 amino acid residues (M(r) of 59,301), which shared weak but significant sequence similarity with various flavoenzymes and contained a nucleotide-binding motif. We found that 2-CAA is converted into pyruvate when the reaction was carried out with purified Caa67(YL) in the presence of FAD and a reducing agent [NAD(P)H or sodium dithionite] under anaerobic conditions. The reducing agent was not stoichiometrically consumed during this reaction, suggesting that FADH(2) is conserved by regeneration in the catalytic cycle. When the reaction was carried out in the presence of H(2)(18)O, [(18)O]pyruvate was produced. These results indicate that Caa67(YL) catalyzes the hydration of 2-CAA to form 2-chloro-2-hydroxypropionate, which is chemically unstable and probably spontaneously dechlorinated to form pyruvate. 2-Bromoacrylate, but not other 2-CAA analogs such as acrylate and methacrylate, served as the substrate of Caa67(YL). Thus, we named this new enzyme 2-haloacrylate hydratase. The enzyme is very unusual in that it requires the reduced form of FAD for hydration, which involves no net change in the redox state of the coenzyme or substrate.

Isotope Signatures Allow Identification of Chemically Cross-Linked Peptides by Mass Spectrometry: A Novel Method to Determine Interresidue Distances in Protein Structures through Cross-Linking

Knowledge of protein structures and protein-protein interactions is essential for understanding of biological processes. Recent advances in protein cross-linking and mass spectrometry (MS) have shown significant potential to contribute to this area. Here we report a novel method to rapidly and accurately identify cross-linked peptides based on their unique isotope signature when digested in the presence of H(2)(18)O. This method overcomes the need for specially synthesized cross-linkers and/or multiple MS runs required by other techniques. We validated our method by performing a "blind" analysis of 5 proteins/complexes of known structure. Side chain repacking calculations using Rosetta show that 17 of our 20 positively identified cross-links fit the published atomic structures. The remaining 3 cross-links are likely due to protein aggregation. The accuracy and rapid throughput of our workflow will advance the use of protein cross-linking in structural biology.

Abstract 5568: Development of serum glycoproteomic profiling technology for the identification of pancreatic cancer biomarkers: simultaneous identification of glycosylation sites and site-specific quantification of glycan structure changes

Abstract Glycosylation is one of the most important and abundant post-translational modifications in nature. Glycoproteins play important roles during molecular and cellular recognition in development, growth, and cellular communication and in particular are involved in cancer progression. Glycoproteins have been used as therapeutic targets and biomarkers for cancer prognosis, diagnosis, and monitoring. As mass spectrometry-based systems biology begins to revolutionize our understanding of biomedical sciences, the ability to efficiently and comprehensively profile glycoproteins in biological samples of interest (body fluids, cell surface, etc.) is critical to many biological and clinical researchers. Here we report a new approach to identify carbohydrate-targeting serum biomarkers, termed isotopic glycosidase elution and labeling on lectin column chromatography (IGEL). It is based on glycan structure-specific enrichment of glycopeptides by lectin-column chromatography and site-directed tagging of N-glycosylation sites by 18O during the elution with N-glycosidase. The N-glycosidase elution in the presence of H218O results in the specific amino acid substitution, asparagine to 18O-asparatic acid residue, with 3-Da increase of the peptide molecular weight. The mass spectrometric identification of this 3-Da increase enables absolutely accurate determination of N-glycosylation sites in complicated samples like serum. Furthermore, the combination of IGEL with label-free quantification system Expressionist enabled us not only to identify N-glycosylation sites accurately but also to quantitatively compare glycan structures on each glycosylation site in a single LC/MS/MS analysis. We applied the IGEL method to the comparative analysis of 18 serum samples from 4 healthy controls, 4 chronic pancreatitis patients, 5 stage-I pancreatic cancer patients, and 5 stage-IV pancreatic cancer patients on the Sambucus sieboldiana agglutinin (SSA) lectin column recognizing alpha 2, 6-linked sialic acid residues. QSTAR-Elite tandem mass spectrometry analysis of SSA-IGEL purified serum samples determined 121 individual N-glycosylation sites captured by the SSA lectin. The further t-test and principal component analysis with Expressionist label-free quantification system revealed that the stage-I pancreatic cancer patients group was statistically distinguishable from normal and pancreatitis groups by the affinity changes of 12 glycosylation sites to SSA lectin. This fact indicated that these glycosylation sites should be potential carbohydrate-targeting biomarkers for pancreatic cancer. Thus our quantitative glycoproteomic technology could be extremely valuable for the identification of carbohydrate biomarkers from various body fluid specimens. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5568.

Cytochrome P450 3A4 regiospecific oxygenation of raloxifene: Evidence for the formation of the di‐quinone methide

While the bioactivation of xenobiotics to toxic intermediates through cytochrome P450 (CYP)‐catalyzed oxygenation reactions is a well recognized process, the production of reactive electrophilic intermediates by P450s through dehydrogenation reactions is not well characterized. Raloxifene is a second generation selective estrogen receptor modulator used for the treatment and prevention of osteoporosis in postmenopausal women. Previous studies have shown CYP3A4‐mediated metabolism of raloxifene produces two hydroxylated metabolites and several glutathione (GSH) adducts. The hydroxylated metabolites and one of the GSH adducts could theoretically be derived from the same dehydrogenated intermediate. It is postulated that raloxifene is dehydrogenated to a reactive di‐quinone methide. To evaluate the formation of the di‐quinone methide, raloxifene was incubated with CYP3A4 in the presence of H218O or 18O2. LC/MS analysis showed that 3′‐hydroxyraloxifene incorporated oxygen only from 18O2, while the source of the oxygen for the 7‐hydroxyraloxifene was inconclusive. These results demonstrate CYP3A4 selectively oxygenates raloxifene from 18O2 at the 3′‐position, but not at the 7‐position. Furthermore, the lack of CYP3A4‐mediated oxygenation at the 7‐position suggests that the di‐quinone methide is hydrated by active‐site H2O, but labeled H218O could not serve as the source of hydration. Supported by NIH grant # GM074249 form the National Institute of General Medical Sciences.

Photo‐oxidative dealkylation of α‐alkylbenzyl methyl ethers induced by titanium dioxide in acetonitrile

AbstractTiO2‐sensitised photo‐oxidation of α‐alkylbenzyl methyl ethers in deaerated CH3CN gives the expected corresponding ketone, whereas in an aerated medium a dealkylation compound (methyl benzoate) is obtained as the principal product, with respect to the ketone, or the exclusive product (when the alkyl group is tert‐butyl). The product analysis and distribution, together with a qualitative estimate of the relative reactivity, suggest that the mechanism in deaerated CH3CN is the same as that previously hypothesized for non‐α‐alkylated benzyl ethers; in particular, the carbonyl compound should be formed through oxidation of the benzylic radical (obtained by deprotonation of the cation radical) to the corresponding cation. To justify the formation of methyl benzoate in an aerated medium, a reasonable hypothesis is that the α‐alkyl‐α‐methoxybenzylperoxy radical (obtained from the competitive attack of oxygen on the benzylic radical) undergoes a dealkylation process; in particular, this intermediate, as a tertiary peroxy radical, could form a dimer that evolves into the corresponding oxy radical, giving the ester through a β‐scission process. The general mechanism suggested in aerated medium was confirmed by evaluating the relationship between the ester/carbonyl (E/C) molar ratio and the adiabatic ionization potential of the benzylic radical intermediate. To evaluate the influence of medium heterogeneity, the E/C ratio data were compared with those obtained from an electron‐transfer photosensitised [by 9,10‐dicyanoanthracene (DCA)] oxidation in a homogeneous phase. A significant confirmation of the mechanism, in both deaerated and aerated media, was obtained by the reaction performed from 4‐methoxy‐α‐ethylbenzyl methyl ether (as a model) in the presence of H218O, sensitised by either TiO2 or DCA. Copyright © 2005 John Wiley & Sons, Ltd.

18O-Tracer Studies of Fe(II)-Induced Decomposition of 1,2,4-Trioxolanes (Ozonides) Derived from Cyclopentenes and Indenes. Inner-Sphere Electron Transfer Reduction of the Peroxide Linkage

Fe(II)-induced decomposition of 1,2,4-trioxolanes (ozonides) 1 and 2 is studied in detail. The inner-sphere electron transfer reduction, which is sensitive to steric effects, is proposed to be a reasonable mechanism for the peroxide decomposition, i.e., the selective generation of one of the two possible Fe(III)-complexed oxy radicals. The fate of the oxy radical species is revealed in detail by using 18O-tracer studies in the reduction of 18O-labeled ozonides 1a and 2a, in which the ethereal oxygens are labeled by 18O atom (18O, 77%) and also by the reduction in the presence of H218O (18O, 10%). The results of the 18O-tracer studies are consistent with the regioselective generation of the Fe(III)-complexed oxy radical species.

Ligand oxidation in N4 tetradentate Schiff base complexes catalyzed by copper(II) hexafluoroacetylacetonate dihydrate: reaction details and structures

The ethylene unit connecting the two anilino nitrogen atoms of the copper or nickel complexes of 7,8,15,16,17,18-hexahydro-dibenzo[e,m][1,4,8,11]tetraazacyclotetradecine [H2(2,2 mac)] and 8,9,16,17,18,19-hexahydro-7H-dibenzo[e,n][1,4,8,12]tetraazacyclopentaadecine [H2(3,2 mac)], i.e. M(2,2 mac) and M(3,2 mac), are oxidized in chloroform at room temperature under atmospheric conditions in the presence of Cu(hfa)2·(H2O)2 to the oxamide (N–CH2–CH2–N– to N–CO–CO–N–). If Cu(hfa)2·(H2O)2 is present in stoichiometric amounts the isolated product of the reaction is the dinuclear complex, M(3,2 oxomac)Cu(hfa)2. The dinuclear complex can also be prepared by direct reaction of M(3,2 oxomac) (prepared by another method) with Cu(hfa)2·(H2O)2. The dinucleation reaction of M(3,2 oxomac) with M(hfa)2 is quite general but the ligand oxidation of M(3,2 mac) is specific for Cu(hfa)2·(H2O)2. Reaction of Cu(3,2 mac) with Hhfa or H2(3,2 mac) with Cu(hfa)2·(H2O)2 results not in oxidation of the macrocycle but in protonation to give Cu{H2(3,2 mac)}(hfa)2. The oxidation of Cu(3,2 mac) in an 18O2 atmosphere does not result in a significant incorporation of 18O. However, nearly quantitative incorporation of 18O is achieved when the reaction is carried out under air in the presence of H218O. The structures of Cu(3,2 mac), Cu(3,2 oxomac), Cu(3,2 oxomac) Cu(hfa)2 and Cu{H2(3,2 mac)}(hfa)2 are reported.

Enzymatic Synthesis of 18O(6)-Guanosine and Spectroscopic Characterization of Hoogsteen and Reverse Hoogsteen Hydrogen-Bonded Guanosine Structures

Adenylic acid deaminase has been successfully used for the first time to convert Cl(6)-guanosine to 18O(6)-guanosine in the presence of H218O. Observations of infrared and Raman spectra of both guanosine and 18O(6)-guanosine permit assignment of the carbonyl vibrations in guanosine tetramer. In-plane normal mode wavenumbers have been calculated using a valence force field that incorporates an interaction force constant of νCO transition dipole coupling. This coupling alone between carbonyl groups of adjacent guanine residues provides the physical basis for understanding the observed CO splittings in guanosine tetramer and in hydrogen-bonded guanine bases adopting the Hoogsteen structure. This coupling is, however, absent in the dimer of 2‘-deoxy-3‘,5‘-bis(triisopropylsilyl)guanosine, which is known to adopt the reverse Hoogsteen structure. The νCO spectral profiles, then, permit the distinction between Hoogsteen and reverse Hoogsteen hydrogen-bonded guanine nucleobases in G*G·C triplets that may be presen...

Novel techniques for identification and characterization of proteins loaded on gels in femtomole amounts.

A combination of techniques is presented allowing gel-purified protein identification in the femtomole range using matrix-assisted-laser-desorption-ionization mass spectrometry. The proteins are detected in the primary gel by a sensitive negative staining procedure, transferred, and concentrated in a secondary gel matrix. There, they are digested in the presence of H218O and their sequences are predicted (1) by peptide mass fingerprinting, (2) by comparing the post-source-decay (PSD) spectra with theoretical spectra of candidate isobaric peptides using a computer algorithm called MassFrag, and (3) by a manual readout of the 18O/16O-labeled fragmentation ions in the PSD spectra.

Determination of Reactive Intermediates in Iron Porphyrin Complex-Catalyzed Oxygenations of Hydrocarbons Using Isotopically Labeled Water: Mechanistic Insights

We have studied iron porphyrin complex-catalyzed oxygenations of hydrocarbons by several oxidants (i.e., hydrogen peroxide, tert-butyl hydroperoxide, and m-chloroperoxybenzoic acid (MCPBA)) in the presence of H218O. In the olefin epoxidation and alkane hydroxylation reactions catalyzed by (meso-tetrakis(pentafluorophenyl)porphinato)iron(III) chloride [Fe(F20TPP)Cl], the percentages of 18O incorporated into the oxygenated products were found to be the same in all of the reactions of hydrogen peroxide, tert-butyl hydroperoxide, and MCPBA, leading us to conclude that a common high-valent iron oxo complex was the reactive intermediate responsible for oxygen atom transfer. When the epoxidation of cyclooctene by MCPBA and H2O2 was performed at low temperature in the presence of H218O, it was found that there was no 18O-incorporation from labeled water into cyclooctene oxide. We interpreted the lack of 18O-incorporation in these reactions with that an electronegatively-substituted iron porphyrin complex forms a relatively stable (Porp)FeIII−OOR species and this intermediate transfers its oxygen to olefin prior to the O−O bond cleavage at low temperature. As the reaction temperature raised from −78 °C to room temperature, the amount of 18O incorporated into the oxide product gradually increased in the reactions of cyclooctene epoxidation. This was attributed to the fast conversion of FeIII−OOR to the high-valent iron oxo complex via the O−O bond cleavage at higher temperature. We found, by studying the effects of the olefin and H218O concentrations on the amount of 18O incorporated into the oxide product, that the rate of the oxygen exchange between high-valent iron oxo complex and labeled water was slower than that of the oxygen atom transfer from the intermediate to organic compounds in catalytic oxygenation reactions. Blocking an axial position of iron porphyrin complex with imidazole prevented the 18O-incorporation from labeled water into the oxygenated products, explaining the phenomenon of no oxygen exchange in cytochrome P-450 systems.

Intracellular Phosphate–Water Oxygen Exchange Measured by Mass Spectrometry

To study,in vivo,potential Pi–water oxygen exchange catalyzed by each of two high-affinity Pisymporters ofNeurospora crassa,we have developed methods for the purification of Pifrom whole-cell extracts and the subsequent derivatization of Pifor analysis by GC–MS. We have also modified a published procedure for the preparation of18O-Pi. However, the high background rate of transport-independent oxygen exchange, determined by monitoring the appearance of18O-Piin cells incubated in the presence of H218O, masks detection of any transport-dependent oxygen exchange which may occur. The rate of intracellular Pi–water oxygen exchange is 4.36 nmol18O incorporated into Piper second per milligram of cell protein. The18O isotopic distribution of the intracellular Piclosely resembles that predicted for random exchange of a single Pioxygen with that of water per enzymatic event. Based upon the observed isotopic enrichment, we calculate that the bulk intracellular Pipool must undergo an average of one oxygen exchange per phosphate ion about every 3 s.

H218O isotope exchange studies on the mechanism of reduction of nitric oxide and nitrite to nitrous oxide by denitrifying bacteria. Evidence for an electrophilic nitrosyl during reduction of nitric oxide

Reduction of NO and NO2-by whole cells of eight strains of denitrifying bacteria known to contain either heme cd1 or copper-containing nitrite reductases (NiRs) has been examined in the presence of H218O. All organisms containing heme cd1 NiRs exhibited relatively large extents of exchange between NO2- and H218O (39-100%), as monitored by the 18O content of product N2O. Organisms containing copper NiRs gave highly variable results, with Achromobacter cycloclastes and Pseudomonas aureofaciens exhibiting no 18O incorporation and Rhodopseudomonas sphaeroides and Alcaligenes entrophus exhibiting complete exchange between NO2- and H218O. Organisms containing heme cd1 NiRs exhibited significant but lower levels of exchange between NO and H218O than between NO2- and H218O, while organisms containing copper NiRs gave significantly higher amounts of 18O incorporation than observed for the heme cd1 organisms. These results demonstrate the existence of an NO-derived species capable of undergoing O-atom exchange with H218O during the reduction of NO. Trapping experiments with 15NO, 14N3-, and crude extracts of R. sphaeroides support the electrophilic nature of this intermediate and suggest its formulation as an enzyme nitrosyl, E-NO+, analogous to that observed during reduction of NO2-. The observation of lower levels of 18O incorporation with NO2- than with NO as substrate for A. cycloclastes and P. aureofaciens indicates that, for these organisms at least, a sequential pathway involving free NO as an intermediate is significantly less important than a direct pathway in which N2O is formed via reaction of two NO2- ions on a single enzyme.

Barley powdery mildew “Invertase” is an alpha-glucosidase

Extracts from barley powdery mildew (Erysiphe graminis hordeii) were found to contain sucrose-hydrolase and α-glucosidase activities with identical pH optima. When fractionated by anion-exchange and size exclusion chromatography, these extracts showed coincidence of the two enzyme activities in the fractions obtained. Experiments were performed in which sucrose was hydrolysed in the presence of H2 18O by yeast invertase (E.C. 3.2.1.26), yeast α-glucosidase (E.C. 3.2.1.20), and by an extract from the mycelium of barley powdery mildew. Using GC/MS, the distribution of18O between derivatives of the hexose products of each reaction was determined. From a comparison of the results, it is concluded that the hydrolysis of sucrose by the powdery mildew extract was catalysed by an α-glucosidase.