O2 incorporation into a long-chain fatty acid during bacterial luminescence

The bioluminescence-dependent oxidation of a long-chain fatty aldehyde catalyzed by luciferase from Photobacterium phosphoreum has been studied in ¹⁸O₂ experiments. The results show the incorporation of one atom of molecular oxygen into the product, the corresponding fatty acid. This incorporation is not the result of exchange of ¹⁸O₂ with the aldehyde prior to oxidation to the acid, thereby indicating that the bacterial luciferase catalyzes an aldehyde monooxygenase reaction which is coupled with bioluminescence.     

Oxygen fixation into hydroxyproline in etiolated maize seedlings : verification by tandem mass spectrometry

Etiolated maize (Zea mays L.) seedlings were grown in the dark for 5 days in an atmosphere enriched with 10.0 atom% ¹⁸O₂. Hydroxyproline was isolated from root and shoot tissues, purified, and methylated. It was not possible to determine ¹⁸O incorporation into hydroxyproline by conventional mass spectrometry because the final product was not sufficiently pure. The final product was analyzed successfully by tandem mass spectrometry. The ¹⁸O content of the hydroxyl oxygen atom was 10 ± 0.7 atom%. This result demonstrates that the hydroxyl oxygen atom in hydroxyproline was derived exclusively from molecular oxygen.     

Spontaneous Hydrolysis and Dehydration of Dehydroascorbic Acid in Aqueous Solution

The interaction of water with dehydroascorbic acid was examined by incubating dehydroascorbic acid and ascorbic acid in¹⁸O-labeled water for various amounts of time and then oxidizing the products with hydrogen peroxide or reducing the products with mercaptoethanol, with analysis by gas chromatography mass spectrometry. Based on mass changes, dehydroascorbic acid readily exchanged three oxygen atoms with H₂¹⁸O. When mercaptoethanol was used to reduce dehydroascorbic acid (which had been incubated in H₂¹⁸O) to ascorbic acid, the newly formed ascorbic acid also contained three labeled oxygen atoms. However, ascorbic acid incubated in H₂¹⁸O for the same amount of time under identical conditions exchanged only two labeled oxygen atoms. Electron impact mass spectrometry of derivatized ascorbic acid created a decarboxylation product which had only two labeled oxygen atoms, regardless if 3-oxygen-labeled or 2-oxygen-labeled ascorbic acid was the parent compound, isolating the extra oxygen addition to carbon 1. These data suggest that dehydroascorbic acid spontaneously hydrolyzes and dehydrates in aqueous solution and that the hydrolytic-hydroxyl oxygen is accepted by carbon 1. Ascorbic acid, on the other hand, does not show this same tendency to hydrolyze.     

Hydroperoxide-dependent oxygenation of trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene by ram seminal vesicle microsomes. Source of the oxygen

Incubation of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid with ram seminal vesicle microsomes (RSVM) triggers the oxygenation of $\text{trans}$-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol). The principal oxidation products are 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrenes which are non-enzymatic hydrolysis products of r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene. At short incubation times, an additional product is isolated which is identified as r-7,t-8,t-9-trihydroxy-c-10-methoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. This product appears to arise by solvolysis of the extracted diolepoxide during high performance liquid chromatography using methanol-water solvent systems. The incubation of ¹⁸O-labeled 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid with BP-7,8-diol and RSVM leads to very little incorporation of ¹⁸O into the stable solvolysis products (analyzed by gc-ms of their peracetates). Parallel incubations conducted with ¹⁶O-labeled hydroperoxide under an ¹⁸O₂ atmosphere indicate that the principle source of the epoxide oxygen is molecular oxygen.     

Utilization of nitrate byBeggiatoa alba

Beggiatoa alba B18LD utilizes both nitrate and nitrite as sole nitrogen sources, although nitrite was toxic above 1 mM.B. alba coupledin vivo acetate oxidation, but not sulfide oxidation, with nitrate and nitrite reduction.B. alba could not, however, grow anaerobically with nitrate as the sole electron acceptor. Furthermore, the incorporation of acetate into macromolecules under anaerobic conditions with nitrate as the sole electron acceptor was less 10% of the incorporation with oxygen as the electron acceptor. The product of nitrate reduction byB. alba was ammonia; N₂ or N₂O were not produced. The nitrate reductase activity inB. alba was soluble and it utilized reduced flavins or methyl viologen and dithionite as electron donors. Pyrimidine nucleotides were not used as in vitro electron donors, either alone or with flavins in coupled assays. TheB. alba nitrate reductase activity was competitively inhibited with chlorate and was only mildly inhibited by azide and cyanide. Nitrate was not required for induction of theB. alba nitrate reductase, and neither oxygen nor ammonia repressed its activity. Thus,B. alba nitrate reductase appears to be an assimilatory nitrate reductase with unusual regulatory properties.Non-standard abbreviations MV Methyl viologen - DT dithionite - GS glutamine synthetase - GOGAT glutamine 2-oxoglutarate aminotransferase - PPO 2-diphenyloxazole - POPOP 1,4-(bis)-[2-(5-phenyloxazolyl)] benzene - TCA trichloroacetic acid - CCCP carbonylcyanidem-chlorophenylhydrazone - FCCP carbonylcyanidep-trifluoromethoxyphenylhydrazone - TTFA thenoyltrifluoroacetone - PHEN 1,10-phenanthroline - HOQNO 2-heptyl 4-hydroxyquinoline-n-oxide - 8HQ 8-hydroxyquinoline     

Source of oxygen in cytochrome P-450 catalyzed carbinolamine formation

N-Methylcarbazole was incubated in H₂O¹⁸ and under an ¹⁸O atmosphere. N-Hydroxymethylcarbazole, 1-OH- and 3-OH-N-methylcarbazole were isolated by HPLC and analyzed for ¹⁸O content In incubations containing ¹⁸O, all three metabolites showed >95% ¹⁸O incorporation. In incubations containing H₂O¹⁸, the N-hydroxymethyl metabolite showed ¹⁶O incorporation equal to control incubations in 100% H₂O. These data demonstrate that the sole source of oxygen in cytochrome P-450 catalyzed, NADPH supported N-hydroxymethylcarbazole formation is atmospheric oxygen.     

Inactivation of Escherichia coli by superoxide radicals and their dismutation products.

Escherichia coli cells are inactivated by the products of the reaction between dialuric acid and oxygen, of which the primary product is Superoxide. The rate of inactivation is decreased by Superoxide dismutase, by catalase, and by EDTA, whereas it is increased by addition of cupric ions or hydrogen peroxide. It is concluded that a toxic product is formed in a reaction involving Superoxide, hydrogen peroxide, and metal ions, which might be the Haber-Weiss reaction, O₂^? + H₂O₂ → OH + OH^? + O₂. In radiation chemical experiments it is shown that this reaction does not occur in the absence of metal ions.     

f-Element/crown ether complexes. 16. Synthesis,crystallization and crystal structure of [Dy(OH2)8]Cl3·18-crown-6·4H2O

When crystals of [Dy(OH₂)₇(OHMe)] [DyCl(OH₂)₂(18- crown-6)]₂Cl₇·2H₂O [1] are allowed to warm from 5 °C to ambient temperature (22 °C) under the original solvent mixture (1:3 CH₃OH: CH₃CN), they redissolve and the title complex can be isolated by slow evaporation of the resulting solution. The crystal structure of this complex, [Dy(OH₂)₈]Cl₃·18-crown-₆·4H₂O, has been determined. It crystallizes in the monoclinic space group, P2₁/c, with a = 10.395(1), b = 18.684(1), c = 16.259- (3) Å, β= 102.56(1)°, and D_calc = 1.61 g cm⁻³ for Z = 4. A final conventional R value of 0.041 was obtained by least-squares refinement using 3453 independent observed [F_o⩾5σ(F_o)] reflections. The [Dy(OH₂)₈]³⁺ cations and crown ether molecules are hydrogen bonded in a polymeric chain with the crown molecules separating the cations and a total of seven DyOH₂···O(crown ether) hydrogen bonds. The chains are connected by a hydrogen bonding network consisting of the cations, chloride ions, and uncoordinated water molecules. The geometry of the cation is best described as a bicapped trigonal prism with distortions on the reaction pathway toward dodecahedral symmetry. The two capping atoms average 2.41(1) Å from Dy, the remaining DyO distances average 2.38(2) Å. The 18-crown-6 molecule has the D_3d conformation normally observed except for a distortion of one OCCO unit containing the oxygen atom accepting two hydrogen bonds.     

Selective changes in fatty acid composition of phosphatidylserine in rat erythrocyte membrane induced by nitrate

The relationship between nitrate which is formed from inhaled nitrogen dioxide, a common air pollutant, and changes in fatty acid metabolism of phosphatidylserine in rat erythrocytes has been examined. When erythrocytes were incubated at 37°C for 60 min with fatty acid, the incorporation rate of [1-¹⁴C]arachidonic acid and [9,10-³H]palmitic acid into phosphatidylserine was 15% (80 pmol/h per μmol lipid phosphorus) and 20% (12 pmol/h per μmol lipid phosphorus) of those into phosphatidylethanolamine, respectively. By the addition of 1.0 mM sodium nitrate or 0.5 μM ionophore A23187 to the incubation mixture, the rate of incorporation of both arachidonic acid and palmitic acid into phosphatidylethanolamine was stimulated 1.45-fold. On the other hand, the incorporation of palmitic acid into phosphatidylserine was little affected, while that of arachidonic acid was stimulated 1.35-fold. An increase in arachidonic acid of phosphatidylserine was also found by the addition of nitrate or ionophore A23187. This increase was dependent on the concentration of extracellular calcium and observed by the addition of other chaotropic anions in the order SCN >CIO₄^? > NO₃^?. It seems likely, therefore, that nitrate causes changes in erythrocyte membranes to facilitate calcium uptake. Increasing the concentration of intracellular calcium may cause stimulation of acyl-CoA:lysophospholipid acyltransferase and/or endogenous phospholipase A₂.     

Trichothecene Biosynthesis in Fusarium sporotrichioides: Origin of the Oxygen Atoms of T-2 Toxin

Mass spectral analysis of T-2 toxin formed during the growth of Fusarium sporotrichioides (ATCC 24043) in the presence of H₂¹⁸O showed incorporation of up to three ¹⁸O atoms per toxin molecule. The carbonyl oxygens of the acetates at C-4 and C-15 and of the isovalerate at C-8 were derived from H₂O. Toxin formed in the presence of ¹⁸O molecular oxygen incorporated up to six ¹⁸O atoms per toxin molecule. The overall incorporation was 78 and 92% of toxin molecules labeled for H₂¹⁸O and ¹⁸O₂ labeled samples, respectively. The oxygens of position 1, the 12,13-epoxide, and the hydroxyl groups at C-3, C-4, C-8, and C-15 were all derived from molecular oxygen.     

Biochemical studies on the lethal effects of solar and artificial ultraviolet radiation on Staphylococcus aureus

The effects of UV-B radiation generated in the laboratory and as a component of sunlight on the viability and particular biochemical activities of the bacterium Staphylococcus aureus have been examined. UV-B radiation progressively inhibits protein synthesis (assayed as ³H-alanine incorporation) and kills cells. Cell respiration, and RNA and DNA synthesis (³H-uridine and ³H-thymidine incorporation) were not greatly affected by UV-B irradiation. The OH and ¹O₂-free radical scavengers protected cells against killing and inhibition of protein synthesis by UV-B, suggesting that such radicals mediate the effects of UV-B on this organism. A similar protective effect using a ferric ion chelator suggests an important role for metallic ions in UV-B lethality.Abbreviations VIS, UV-A, UV-B, UV-C radiation in the bands 400–750 nm, 315–400 nm, 280–315 nm, 200–280 nm respectively - DBCO diazabicyclooctane - OFR oxygen free radical - OH, ¹O₂, O _inf2 ^sup- hydroxyl free radical, singlet oxygen, superoxide radical respectively     

Modeling the scavenging activity of ellagic acid and its methyl derivatives towards hydroxyl, methoxy, and nitrogen dioxide radicals

The reaction mechanisms involved in the scavenging of hydroxyl (OH^·), methoxy (OCH₃ ^·), and nitrogen dioxide (NO₂ ^·) radicals by ellagic acid and its monomethyl and dimethyl derivatives were investigated using the transition state theory and density functional theory. The calculated Gibbs barrier energies associated with the abstraction of hydrogen from the hydroxyl groups of ellagic acid and its monomethyl and dimethyl derivatives by an OH· radical in aqueous media were all found to be negative. When NO₂ ^· was the radical involved in hydrogen abstraction, the Gibbs barrier energies were much larger than those calculated when the OH^· radical was involved. When OCH₃ ^· was the hydrogen-abstracting radical, the Gibbs barrier energies lay between those obtained with OH^· and NO₂ ^· radicals. Therefore, the scavenging efficiencies of ellagic acid and its monomethyl and dimethyl derivatives towards the three radicals decrease in the order OH^· >> OCH₃ ^· > NO₂ ^·. Our calculated rate constants are broadly in agreement with those obtained experimentally for hydrogen abstraction reactions of ellagic acid with OH· and NO₂· radicals.

Catabolism of Arylboronic Acids by Arthrobacter nicotinovorans Strain PBA

Arthrobacter sp. strain PBA metabolized phenylboronic acid to phenol. The oxygen atom in phenol was shown to be derived from the atmosphere using ¹⁸O₂. 1-Naphthalene-, 2-naphthalene-, 3-cyanophenyl-, 2,5-fluorophenyl-, and 3-thiophene-boronic acids were also transformed to monooxygenated products. The oxygen atom in the product was bonded to the ring carbon atom originally bearing the boronic acid substituent with all the substrates tested.     

Nitrogen Nutrition and Xylem Sap Composition of Peanut (Arachis hypogaea L. cv Virginia Bunch)

The principal forms of amino nitrogen transported in xylem were studied in nodulated and non-nodulated peanut (Arachis hypogaea L.). In symbiotic plants, asparagine and the nonprotein amino acid, 4-methyleneglutamine, were identified as the major components of xylem exudate collected from root systems decapitated below the lowest nodule or above the nodulated zone. Sap bleeding from detached nodules carried 80% of its nitrogen as asparagine and less than 1% as 4-methyleneglutamine. Pulse-feeding nodulated roots with ¹⁵N₂ gas showed asparagine to be the principal nitrogen product exported from N₂-fixing nodules. Maintaining root systems in an N₂-deficient (argon:oxygen, 80:20, v/v) atmosphere for 3 days greatly depleted asparagine levels in nodules. 4-Methyleneglutamine represented 73% of the total amino nitrogen in the xylem sap of non-nodulated plants grown on nitrogen-free nutrients, but relative levels of this compound decreased and asparagine increased when nitrate was supplied. The presence of 4-methyleneglutamine in xylem exudate did not appear to be associated with either N₂ fixation or nitrate assimilation, and an origin from cotyledon nitrogen was suggested from study of changes in amount of the compound in tissue amino acid pools and in root bleeding xylem sap following germination. Changes in xylem sap composition were studied in nodulated plants receiving a range of levels of ¹⁵N-nitrate, and a ¹⁵N dilution technique was used to determine the proportions of accumulated plant nitrogen derived from N₂ or fed nitrate. The abundance of asparagine in xylem sap and the ratio of asparagine:nitrate fell, while the ratio of nitrate:total amino acid rose as plants derived less of their organic nitrogen from N₂. Assays based on xylem sap composition are suggested as a means of determining the relative extents to which N₂ and nitrate are being used in peanuts.     

Effect of Glucose and CO(2) on Nitrate Uptake and Coupled OH Flux in Ankistrodesmus braunii

In Ankistrodesmus braunii, in the absence of CO₂, i.e. in CO₂-free air or N₂, photosynthetic nitrate uptake and nitrate reduction were inhibited, especially at low pH. Under such conditions, glucose stimulated nitrate uptake and reduction to almost the same level in the pH range between 6 and 8.5. CO₂ at 0.03% effected an intermediate pH dependence of nitrate uptake; saturating CO₂ concentration (more than 1%) eliminated the pH dependence, as did glucose, but the rates were enhanced compared with glucose. Glucose and, even more, CO₂, drastically reduced the release of nitrite and ammonia to the medium, the stoichiometry between alkalinization of the medium and nitrate uptake (OH⁻/NO₃⁻) approached 1.     

Evidence for a universal pathway of abscisic Acid biosynthesis in higher plants from o incorporation patterns

Previous labeling studies of abscisic acid (ABA) with ¹⁸O₂ have been mainly conducted with water-stressed leaves. In this study, ¹⁸O incorporation into ABA of stressed leaves of various species was compared with ¹⁸O labeling of ABA of turgid leaves and of fruit tissue in different stages of ripening. In stressed leaves of all six species investigated, avocado (Persea americana), barley (Hordeum vulgare), bean (Phaseolus vulgaris), cocklebur (Xanthium strumarium), spinach (Spinacia oleracea), and tobacco (Nicotiana tabacum), ¹⁸O was most abundant in the carboxyl group, whereas incorporation of a second and third ¹⁸O in the oxygen atoms on the ring of ABA was much less prominent after 24 h in ¹⁸O₂. ABA from turgid bean leaves showed significant ¹⁸O incorporation, again with highest ¹⁸O enrichment in the carboxyl group. The ¹⁸O-labeling pattern of ABA from unripe avocado mesocarp was similar to that of stressed leaves, but in ripe fruits there was, besides high ¹⁸O enrichment in the carboxyl group, also much additional ¹⁸O incorporation in the ring. In ripening apple fruit tissue (Malus domestica), singly labeled ABA was most abundant with more ¹⁸O incorporated in the tertiary hydroxyl group than in the carboxyl group of ABA. Smaller quantities of this monolabeled product (C-1′-¹⁸OH) were also detected in the stressed leaves of barley, bean, and tobacco, and in avocado fruits. It is postulated that a large precursor molecule yields an aldehyde cleavage product that is, in some tissues, rapidly converted to ABA with retention of ¹⁸O in the carboxyl group, whereas in ripening fruits and in the stressed leaves of some species the biosynthesis of ABA occurs at a slower rate, allowing this intermediate to exchange ¹⁸O with water. On the basis of ¹⁸O-labeling patterns observed in ABA from different tissues it is concluded that, despite variations in precursor pool sizes and intermediate turnover rates, there is a universal pathway of ABA biosynthesis in higher plants which involves cleavage of a larger precursor molecule, presumably an oxygenated carotenoid.     

Identification of nitrogen sources and transformations within karst springs using isotope tracers of nitrogen

Isotope analyses of nitrate and algae were used to gain better understanding of sources of nitrate to Florida’s karst springs and processes affecting nitrate in the Floridan aquifer at multiple scales. In wet years, δ¹⁵N and δ¹⁸O of nitrate ranged from +3 to +9‰ in headwater springs in north Florida, indicating nitrification of soil ammonium as the dominant source. With below normal rainfall, the δ¹⁵N and δ¹⁸O of nitrate were higher in almost all springs (reaching +20.2 and +15.3‰, respectively) and were negatively correlated with dissolved oxygen. In springs with values of δ¹⁵N-NO₃ and δ¹⁸O-NO₃ greater than +10‰, nitrate concentrations declined 40–50% in dry years and variations in the δ¹⁵N and δ¹⁸O of nitrate were consistent with the effects of denitrification. Modeling of the aquifer as a closed system yielded in situ fractionation caused by denitrification of 9 and 18‰ for Δ¹⁸O and Δ¹⁵N, respectively. We observed no strong evidence for local sources of nitrate along spring runs; concentrations declined downstream (0.42–3.3?μmol-NO₃ L^?1 per km) and the isotopic dynamics of algae and nitrate indicated a closed system. Correlation between the δ¹⁵N composition of nitrate and algae was observed at regional and spring-run scales, but the relationship was complicated by varying isotopic fractionation factors associated with nitrate uptake (Δ ranged from 2 to 13‰). Our study demonstrates that nitrate inputs to Florida’s springs are derived predominantly from non-point sources and that denitrification is detectable in aquifer waters with relatively long residence time (i.e., matrix flow).     

Effects of oxygen on Pseudomonas nautica growth on n-alkane with or without nitrate

The denitrifying marine bacterium, Pseudomonas nautica 617, can grow on lactate aerobically or anaerobically in presence of nitrate with generation times of 1.5 and 3 h respectively. The growth on heptadecane occurs only in presence of oxygen whatever its concentration with a genrration time of 8.5 h. The influence of oxygen, carbon sources (lactate or heptadecane) and nitrate was examined on O₂, NO₃ ^-, NO₂ ^- consumption, on nitrate and nitrite reductases activities, on cell yields, and on the ratio of CO₂ produced per unit of biomass. Pseudomonas nautica metabolizes hydrocarbons under denitrifying conditions in the presence of oxygen. Nitrate and nitrite are used during growth on lactate and heptadecane up to oxygen concentrations corresponding to 50 and 30% of air-saturation, respectively. When growth on n-alkane was not oxygen-limited (above 50% of air-saturation) the catabolism decreases in favour of carbon incorporation into the cell. Nitrate and nitrite reductases were strongly inhibited after 20% of airsaturation in the presence of lactate as growth substrate. With n-alkane, only the nitrate reductase activity was greatly reduced.     

Possible Mechanism of Oxygen Activation in Linoleate Peroxidation by Fusarium Lipoxygenase

The Oxygen activating mechanism of Fusarium lipoxygenase, a heme-containing dioxygenase, was studied. The enzyme did not require any cofactors, such as H₂O₂, however, both superoxide dismutase and catalase inhibited linoleate peroxidation by Fusarium lipoxygenase. A low concentration of H₂O₂ caused a distinct acceleration in enzymatic peroxidation. These results indicate that both O₂? and H₂O₂ are produced as essential intermediates of oxygen activation during formation of linoleate hydroperoxides by Fusarium lipoxygenase. This peroxidation reaction was also prevented by scavengers of singlet oxygen (¹O₂), but not by scavengers of hydroxy 1 radical (OH). Generation of O₂? in the enzyme reaction was detected by its ability to oxidize epinephrine to adrenochrome. Moreover, the rate of peroxide formation was greater in the D₂O than in the H₂O buffer system. These results suggest that the Haber–Weiss reaction (O₂^?+H₂O₂→OH^?+OH^·+¹O₂) is taking part in linoleate peroxidation by Fusarium lipoxygenase, and the ¹O₂ evolved could be responsible for the peroxidation of linoleate. H₂O₂ produced endogenously in the enzyme reaction might act as an activating factor for the enzyme. This possible mechanism of oxygen activation can explain the absence of a need for exogenous cofactors with Fusarium lipoxygenase in contrast to an other heme-containing dioxygenase, tryptophan pyrrolase, which requires an exogenous activating factor, such as H₂O₂.     

Kinetics of the reaction between hydrogen selenide ion and oxygen

Hydrogen selenide ion (HSe^?) reacts with oxygen in the following manner: HSe^? + 1/2O₂ → Se^o + OH^?. Interest in the kinetics of this reaction comes from the fact that selenide is an important product in the metabolism of the essential trace element selenium. Using polarography to monitor both selenide and oxygen, we have found the reaction exhibits complex kinetics, including autoaccelerating behavior and the generation of reactive intermediates capable of inducing reactions in other substances present. Probable intermediate species include superoxide, peroxide and polyselenides. The reaction is slow with respect to diffusion controlled reactions, but fast with respect to the time required to prepare solutions for biological study. Selenide concentrations greater than 10^?6 M decay to give solutions of predominantly colloidal elemental selenium less than 3 minutes after exposure to atmospheric levels of oxygen.