Rapid Reoxidation Research Articles

ChemInform Abstract: SYNTHESIS OF 5‐SELENIUM‐SUBSTITUTED URACIL DERIVATIVES. INHIBITION OF THYMIDYLATE SYNTHETASE BY 5‐HYDROSELENO‐2′‐DEOXYURIDYLATE

AbstractDurch Addition von Methylhypobromit an die Verbindungen (I) und anschließende Umsetzung mit Natriumdiselenid entstehen die Selenverbindungen (III).

Synthesis of 5-selenium-substituted uracil derivatives. Inhibition of thymidylate synthetase by 5-hydroseleno-2'-deoxyuridylate.

5-Selenium-substituted derivatives (diselenides) or uracil, 2'-deoxyuridine, and 2'-deoxyuridylic acid were synthesized via the addition of methyl hypobromite to the 5,6 double bond, followed by reaction of the adducts with sodium diselenide. The physical and chemical properties of these compounds (including their facile reduction by dithiothreitol and rapid reoxidation) were similar to those of the corresponding 5-sulfur analogues. 5-Hydroseleno-2'-deoxyuridylic acid was as potent as 5-mercapto-2'-deoxyuridylate in inhibiting thymidylate synthetase from L. casei (ki approximately 6 X 10(-8) M) but the nucleoside III was considerably less active than 5-mercapto-2'-deoxyuridine in the inhibition of growth of the leukemia L1210 cell in culture.

Oxidation of heme proteins by copper(II) complexes. Rates and mechanism of the copper catalysed autoxidation of cytochrome c, myoglobin and hemoglobin

The mechanism of the autoxidation of ferrocytochrome c catalysed by some copper(II) complexes has been estblished. In most cases the rate determining step was found to be the one electron oxidation of the cytochrome by the copper(II) species, which was followed by the rapid reoxidation of the resulting Cu(I) complexes by molecular oxygen. The rate constants for the electron transfer reactions of several copper(II) complexes with ferrocytochrome c, myoglobin and hemoglobin have been measured at 25 °C. It was shown that these rates for the copper complexes, as well as those for several other oxidants, are consistent with simple outer sphere electron transfer through the heme edge for all the heme proteins. the redox potential of the reacting species was found to be the dominant factor in determining the relative rate of the electron transfer reactions. This enables the catalytic activity of different copper complexes to be predicted.

Refolding of reduced, denatured trypsinogen and trypsin immobilized on Agarose beads.

The reoxidation of fully reduced and denatured bovine trypsinogen and the regeneration of the native structure can be accomplished if the protein is initially attached to Agarose beads. Reoxidation was performed under aerobic conditions, in the presence of mercaptoethanol and dehydroascorbate or with a mixture of reduced and oxidized glutathione. In 24 hours, the yields of regenerated trypsinogen were 60 to 70% with 0.2 to 0.6 mg of protein bound/ml of gel but 30% or less if greater than 1.7 mg of protein were bound. Rapid reoxidation, with dehydroascorbate as catalyst, gave molecules which could not be converted to active trypsin. However, if the incorrectly folded structures were placed in a mixture of reduced and oxidized glutathione, the molecules underwent disulfide interchange and could continue to refold. The rapidly reoxidized molecules regained their native structure with the same rate and to the same extent as they did initially in the absence of rapid reoxidation. Therefore, the rate-limiting step in the refolding of trypsinogen was disulfide interchange. The regenerated Agarose-bound trypsinogen displayed the usual properties of the native molecule in (a) its conversion to active trypsin by a process of limited proteolysis, (b) the kinetic constants of the activated product toward typical trypsin substrates, and (c) the limited cleavage of 1 disulfide bond with sodium borohydride. Refoldind of immobilized trypsin was also observed with an overall yield of 50%. Trypsin can fold spontaneously to its native structure even though it lacks the NH2-terminal hexapeptide of its precursor.

Antimycin-insensitive mutants of Candida utilis. II. The effects of antimycin on cytochrome b

1. Cytochrome b-562 is more reduced in submitochondrial particles of mutant 28 during the aerobic steady-state respiration with succinate than in particles of the wild type. When anaerobiosis is reached, the reduction of cytochrome b is preceded by a rapid reoxidation in the mutant. A similar reoxidation is observed in the wild type in the presence of low concentrations of antimycin. 2. In contrast to the wild type, inhibition of electron transport in the mutant has a much higher antimycin titre than effects on cytochromes b (viz., aerobic steadystate reduction; reduction in the presence of substrate, cyanide and oxygen; the ‘red shift’ and lowering of E′ 0 of cytochrome b-562). Moreover, the titration curve of electron transport is hyperbolic whereas the curves for the reduction are sigmoidal. The conclusion is, that in both mutant and wild type, the actions of antimycin on electron transport and cytochromes b are separable. 3. The red shift in the mutant is more extensive than in the wild type. 4. Cytochrome b-558 and cytochrome b-566 (that absorbs in mutant and wild type at 564.5 nm) do not respond simultaneously to addition of antimycin, indicating that they are two separate cytochromes. 5. The difference between the effect of antimycin on electron transport and cytochromes b reduction is also found in intact cells of the mutant. 6. A model is suggested for the wild-type respiratory chain in which (i) the cytochromes b lie, in an uncoupled system, out of the main electron-transfer chain, (ii) antimycin induces a conformation change in QH 2-cytochrome c reductase resulting in effects on cytochrome b and inhibition of electron transport, (iii) a second antimycinbinding site with low affinity to the antibiotic is present, capable of inhibiting electron transport.

Studies on the mechanism of Mycobacterium smegmatis L-lactate oxidase. 5-Deazaflavin mononucleotide as a coenzyme analogue.

Apo-lactic oxidase from Mycobacterium smegmatis reconstituted with the deazaisoalloxazine analogue of FMN, 5-deazaFMN, undergoes reduction by L-lactate but not catalytic reoxidation by O2. The rate of deazaFMN-holo-enzyme reduction by substrate is 1.1 min-1. With L-[alpha-3-H]-lactate, direct tritium transfer to enzyme-bound deazaFMN occurs during reduction. No evidence for a stable covalent lactate-deazaFMN adduct has been obtained. The deaza-FMNH2-enzyme is reoxidized extremely slowly by O2, consistent with the sluggish nonenzymatic reaction of deaza-FMNH2 with oxygen. On the other hand, addition of pyruvate to the deazaFMNH2-enzyme causes rapid reoxidation, a process not detected in the absence of enzyme.

Thiosulfate- and sulfide-dependent pyridine nucleotide reduction and gluconeogenesis in intact Thiobacillus neapolitanus.

Nicotinamide adenine dinucleotide phosphate (reduced form) is formed more rapidly after the addition of thiosulfate to suspensions of intact Thiobacillus neapolitanus in the absence of CO(2) than nicotinamide adenine dinucleotide (reduced form). Measurement of acid-stable metabolites shows this phenomenon to be the result of rapid reoxidation of nicotinamide adenine dinucleotide (reduced form) by 3-phosphoglyceric acid and other oxidized intermediates, which are converted to triose and hexose phosphates, and that, in reality, the rate of nicotinamide adenine dinucleotide (oxidized form) reduction exceeds that of nicotinamide adenine dinucleotide phosphate (oxidized form) by approximately 4.5-fold. The overall rate of pyridine nucleotide reduction by thiosulfate (264 nmol per min per mg of protein) is in excess of that rate needed to sustain growth. Pyridine nucleotide reduction, adenosine triphosphate synthesis, and carbohydrate synthesis are prevented by the uncoupler m-Cl-Carbonylcyanide phenylhydrazone. Sodium amytal inhibits pyridine nucleotide reduction and carbohydrate synthesis are prevented by the uncoupler m-Cl-carbonylcyanide observations are reproduced when sulfide serves as the substrate. The rate of pyridine nucleotide anaerobic reduction with endogenous substrates or thiosulfate is less than 1% of the aerobic rate with thiosulfate. We conclude that the principal, if not the only, pathway of pyridine nucleotide reduction proceeds through an energy-dependent and amytal-sensitive step when either thiosulfate or sulfide is used as the substrate.

The surface reduction of lunar fines, 14163,111

Exposure of Lunar fines (14163,111) to atomic hydrogen resulted in a surface reduction, the final surface density of the added hydrogen being 2 hydrogen atoms/100 Å 2. Exposure to oxygen at 25°C produced a partial reoxidation of the surface while nitrogen had no effect. It is postulated that the solar wind will completely reduce materials on the lunar surface but that exposure to a terrestrial or similar atmosphere will result in rapid partial reoxidation.

γ Radiolysis of Gaseous Carbon Dioxide at High Densities

The γ radiolysis of gaseous carbon dioxide has been investigated at 50 ± 1°C over the density range of 0.2 to 0.6 g/ml. At density of around 0.5 g/ml, G(CO) from pure carbon dioxide showed a very marked increase. Addition of SF6 at these densities efficiently reduced G(CO). From this it appears that formation of carbon monoxide results mostly from charge neutralization. Examination of the mechanisms in the radiolysis of carbon dioxide in gas and liquid phase, and a calculation on a simple model indicate that this neutralization is of geminate character. Upon addition of hydrocarbons which react with oxygen atoms as well as the addition of both hydrocarbons and SF6, G(CO) passed through a maximum and reached the value of 2.2 at higher concentrations of hydrocarbons, independent of density. It was interpreted that (1) the positive ions involved in neutralization are probably clustered CO2 + in agreement with the above inference, and that (2) there are at least two species responsible for the rapid re-oxidation of CO.

The Glucose Oxidase Mechanism: INTERPRETATION OF THE pH DEPENDENCE

Abstract The pH dependence of the steady state parameters of the glucose oxidase (EC 1.1.3.4, from Aspergillus niger) reaction was determined by O2-monitored experiments over the entire pH range from 3 to 10 at 25°, with d-glucose as substrate. The data were fitted to a three-parameter steady state rate equation and the significance of the steady state parameters was examined by stopped flow half-reaction and turnover measurements at the extremes of the pH range used. The major conclusions from these studies can be summarized as follows. 1. At low pH, in the presence of halide, the maximum turn-over number (kcat) is determined entirely by the rate of flavin reduction (k2) in the reductive half-reaction. Furthermore, substrate combines only with an unprotonated form of the oxidized enzyme and the reductive half-reaction can be represented as follows. H+ E0 (K1)/⇄/(H+) E0 + S (k1)/⇄/(k-1) E0 - S (k2)/→ Er + δ-lactone Since kcat and k2 are both specifically decreased by halides at low pH values, it is probable that the turnover rate in the low pH range is also limited by k2 in the absence of halide. The steady state absorption spectrum of E0 - S is indistinguishable from the spectrum of E0. This finding, together with the fact that removal of the 1-hydrogen from d-glucose is a ratelimiting process in flavin reduction is consistent with both a hydride transfer mechanism and with a flavin-glucose adduct mechanism in which this adduct is relatively unstable and never accumulates significantly as a kinetic intermediate. 2. The importance of k2 as a limiting first order process in turnover diminishes as the pH is raised. Thus, at pH 10 the major first order process in turnover is the breakdown of a species of oxidized enzyme, E'0, in the oxidative half-reaction. The rate of this process at pH 10.0 is 214 sec-1, whereas k2 has a value of 800 sec-1. 3. The reduced enzyme exists in two kinetically significant states of ionization, Er and Er-. The rapid reoxidation of Er with O2, to regenerate E0, is predominant at pH values less than 7. At pH values greater than 7, a much less rapid reaction of Er- with O2, leading to the formation of E'0-, becomes increasingly important. The species E'0- is unreactive with glucose and it is the conversion of a protonated form of E'0- to E0 which principally governs kcat at pH values greater than 7. We present a complete kinetic scheme describing the effects of pH and discuss the possible chemical significance of the species E'0.

Effect of ATP on the redox cycle of respiratory chain-linked DPNH dehydrogenase and the localization of coupling site I

On adding DPNH to a phosphorylating membrane preparation in the presence of O 2 the reduction of a chromophore, which has been tentatively identified as nonheme iron of DPNH dehydrogenase, may be observed by dual wavelength spectrophotometry at 470–500 mμ. With rotenone or piericidin present a part of the chromophore remains permanently bleached even after exhaustion of the DPNH. ATP, however, causes rapid and complete reoxidation of the chromophore. This effect is abolished by oligomycin and dinitrophenol. Control experiments show that under these conditions the rotenone block between DPNH dehydrogenase and cytochrome b is substantially complete. Analysis of the data suggest that coupling site I is located between the specific binding site of rotenone and the nonheme irons responsible for the g=1.94 signal, both of which appear to be constituents of DPNH dehydrogenase.

Effect of chronic intake of ethanol on pyridine nucleotide levels in rat liver and kidney.

Adult male Wistar rats were given ethanol in dosage forms providing either 10–12 g/kg per day for 2 weeks or 9–10 g/kg per day for 6 weeks. Pair-fed controls received equicaloric amounts of either powdered chow or sucrose in place of the alcohol. After 2 weeks, ethanol-treated rats showed a significant increase in hepatic NADH2 concentration, a corresponding reduction in NAD concentration, and a fall in the NAD:NADH2 ratio. NADP and NADPH2 were not affected. After 6 weeks, total hepatic NAD + NADH2 in ethanol-treated rats was increased, but the NADH2 concentration remained elevated and the NAD:NADH2 ratio depressed. No similar changes were noted in the kidney. When ethanol was withheld for 16 h before sacrifice, the total NAD + NADH2 remained elevated but the NAD:NADH2 ratio returned to normal. An elevated hepatic concentration of NADPH2 and a reduced NADP: NADPH2 ratio were also found after 6 weeks of ethanol treatment, but these changes were not reversed after 16 h withdrawal of ethanol. The administration of a test dose of ethanol (1 g/kg), after a 16-h withdrawal period, was followed by very similar changes in the hepatic NAD:NADH2 ratio 1 and 4 h later, in ethanol-treated and control rats. Chronic ethanol treatment does not appear to produce adaptive changes leading to a more rapid reoxidation of NADH2.

The chemistry of flavins and flavoproteins: aerobic photochemistry.

1. When a mixture of FMN and a reducing substrate (e.g. unprotonated amine) is illuminated oxygen is consumed. 2. The rate of oxygen uptake increases as oxygen concentration falls with some substrates (type I reaction), but with other substrates (typically aromatic compounds) the rate falls as the oxygen concentration falls (type II reaction). 3. The kinetics of type I reactions with EDTA, dl-alpha-phenylglycine and diethanolamine are all consistent with a mechanism in which the rate-determining step, hydrogen abstraction by the FMN triplet, is followed by rapid reoxidation of reduced FMN by oxygen. The reaction is faster at low oxygen concentrations because oxygen quenches the triplet. 4. The sensitivity of reaction rates to substituents in dl-alpha-phenylglycine can be described by a Hammett rho value of -0.6. 5. Individual rate constants for quenching and reaction of the FMN triplet with substrate were calculated (2.4x10(8) and 2.1x10(7)m(-1)s(-1) respectively for EDTA) on the assumption that oxygen quenches the triplet in a diffusion-controlled reaction. 6. The pH-dependences of oxygen uptake rates with six natural amino acids as substrates were measured. 7. Photoinactivations of l-glutamate dehydrogenase and d-amino acid oxidase by FMN were demonstrated.

Studies of the heme components of cytochrome c oxidase by EPR spectroscopy

Cytochrome c oxidase was studied by electron paramagnetic resonance (EPR) spectroscopy at different states of oxidation in the presence or absence of specific ligands. The EPR signals observed are described, and an attempt is made to quantitatively evaluate and assign them to components of cytochrome c oxidase. According to this only cytochrome a and part of the copper were detected by EPR in the oxidized form of the enzyme. The reduced form was devoid of significant signals. At intermediate states of oxidation, cytochrome a 3 was manifest in its ferric form by a signal typical of high-spin ferric iron. This indicates that in the oxidized form of the enzyme, ferricytochrome a 3 interacted with another component, viz. another molecule of ferricytochrome a 3 or the EPR-undetectable fraction of the copper, so as to result in a decreased paramagnetism of the system. Strong ligands such as cyanide or azide prevent the intermediate appearance of cytochrome a 3 in the high-spin state; in the presence of azide, a low-spin ferric intermediate was detected. The changes in absorbance at 605 mμ and of the EPR signals during complete titrations of the enzyme with NADH and phenazine methosulfate or dithionite have been measured. Whereas the absorbance at 605 mμ measured at room temperature appeared to decrease linearly with added reductant, the EPR-detectable copper was not reduced initially except in the presence of cyanide. Cytochrome a was reduced in a linear fashion, while the EPR-detectable form of ferricytochrome a 3 accumulated and was reduced after cytochrome a. In the presence of azide, an EPR-detectable low-spin form of ferricytochrome a 3 accumulated, which was not readily reducible. The high-spin form of ferricytochrome a 3 was also seen on rapid (> 10 msec) reduction of cytochrome c oxidase by ferricytochrome c or dithionite; it was not seen (> 10 msec) on rapid reoxidation of fully reduced cytochrome c oxidase. These findings support the concept of the separate existence of cytochromes a and a 3, and indicate that cytochrome a and the EPR-undetectable copper are titrated first, at which point ferricytochrome a 3 becomes detectable by EPR. The EPR-detectable copper and cytochrome a 3 are titrated thereafter.

The Bacterial Oxidation of Vitamin B6: VII. Purification, Properties, and Mechanism of Action of an Oxygenase which Cleaves The 3-Hydroxypyridine Ring

Abstract Purification of an inducible oxygenase that opens the pyridine ring during the metabolic degradation of Vitamin B6 by a soil bacterium is described. The enzyme is shown to catalyze Reaction 1, and is readily assayed by following the decrease in absorbance at 340 mµ resulting from utilization of DPNH and disappearance of the hydroxypyridine. The [see PDF for equation] crystalline oxygenase is homogeneous by disc gel electrophoresis and ultracentrifugation, has a sedimentation coefficient (s020, w) of 7.3 S and a molecular weight (by sedimentation equilibrium) of 166,000, and contains 2 moles of FAD per mole. Activity is lost on resolution with acidic ammonium sulfate and can be completely restored with FAD; FMN is inactive. Crystalline preparations of constant FAD content vary in activity from 90 to 300 arbitrary units per mg for reasons still unknown. The oxygenase is highly sensitive to oxidation and to sulfhydryl reagents, but not to chelating agents, and is stabilized by high concentrations of mercaptoethanol (0.1%) and glycerol (50%). Under anaerobic conditions, the FAD-enzyme is reduced to FADH2-enzyme by DPNH, NaBH4, or Na2S2O4; admission of oxygen reoxidizes the enzyme slowly, whereas on addition of oxygen and Compound I rapid reoxidation of the enzyme occurs and the open chain product appears. No spectrophotometrically detectable intermediate is observed either anaerobically or aerobically, and the available evidence indicates that the reaction proceeds in a concerted fashion via a ternary complex of oxygenase, DPNH, and Compound I. When Reaction 1 is conducted in the presence of 18O2, the isotope is incorporated predominantly into the acetyl group, but also into the newly formed carboxyl group of the product. Thus, the enzyme is best classified as a dioxygenase, although some 18O from H218O also is incorporated into the product. A reaction mechanism consistent with these results is presented which also accounts for the deviation of this labeling pattern from that observed with dioxygenases which cleave aromatic rings without concomitant reduction. TPNH is almost as effective as DPNH in Reaction 1. Of several pyridine analogues tested, only 5-pyridoxic acid replaces Compound I as a substrate; it is very much less active in this capacity. An apparently similar oxygenase from another pseudomonad cleaves 5-pyridoxic acid in preference to Compound I, and is specific for TPNH as the second substrate.

Mechanisms of interchange reactions of halogenoplatinate(IV) complexes: the trans-dihalogenobisoxalatoplatinate(IV)–iodide reactions

The kinetics of replacement of X– in trans-Pt ox2X22–(ox = C2O4, X = Cl or Br) by iodide have been studied, and the equilibrium constant for the reduction of the di-iodo-complex by iodide has been measured. The reactions are first order with respect to both complex and iodide ions, and they are considered to go by a rate-determining reduction to Pt ox22–, X–, and XI, followed by a rapid re-oxidation to form the di-iodo-complex: k25°(X = Cl, µ= 0·05M)= 16·8 × 10–2 l. mole–1 sec.–1; k225°(X = Br, µ= 0·025M)= 5·13 × 103 l. mole–1 sec.–1; ΔH‡(X = Cl)= 2·33 ± 0·17 kcal. mole–1; ΔH‡(X = Br)= 4·79 ± 0·31 kcal. mole–1; ΔS‡(X = Cl)=–54·3 ± 0·6 cal. mole–1 deg.–1; ΔS‡(X = Br)=–25·5 ± 1·2 cal. mole–1 deg.–1.The reaction of iodide with the bromo-complex fits into a pattern, established by reactions of iodine with related halogeno-platinate(IV) complexes, in which the rates of reduction appear to be paralleled by the thermodynamic ease of reduction. However, all of the rate parameters for the reaction with the chloro-complex are exceptional

Paper 20: Stability of Self-Generated Oxide Films on Unlubricated En 1A Steel Surfaces

Previous work has shown that within the range 20 < N1/2 U < 600 lbf1,2ft/s friction and wear between unlubricated En 1A steel surfaces is determined primarily by the condition of self-generated oxide films. The stability of these films has been examined using a contact resistance technique and metallurgical examination. At a sliding velocity of 100 ft/s, periodic changes have been observed to occur in the contact resistance between the surfaces. During a cycle the contact resistance increases rapidly at first and then more gradually until a critical value is attained when the film disintegrates. Simultaneous with the surface breakdown there is an increase in the coefficient of friction, pin wear rate, and bulk surface temperature. Conditions are such that rapid re-oxidation occurs, resulting in the formation of a new film A metallurgical examination has shown that even using pin specimens of cold-drawn low carbon steel, subsurface grain re-orientation can be responsible for pearlite-rich layers in the stratified structure produced; hence the hardenability of the substrate, and therefore the support provided for the oxide film, can undergo significant changes as wear proceeds. It is concluded that the unstable nature of self-generated oxide films on steel surfaces is responsible for the periodic increases in friction and wear which have been observed.

Spectral Studies on the Catalytic Mechanism and Activation of Pseudomonas Tryptophan Oxygenase (Tryptophan Pyrrolase)

1. The substrate, tryptophan, forms spectrophotometrically detectable enzyme-substrate complexes with tryptophan oxygenase (EC 1.13.1.12)—commonly known as tryptophan pyrrolase—when the iron atom of the heme prosthetic group is either divalent, trivalent, or combined with carbon monoxide. 2. One-half of the enzyme-bound ferriprotoporphyrin IX undergoes a rapid cyclic valence change during catalysis owing to its sequential reduction by tryptophan and reoxidation by oxygen. During steady state catalysis rapid reoxidation maintains the enzyme predominantly as ferriprotoporphyrin, but addition of CO to the steady state catalytic system leads to the detection and trapping of the reduced form as the CO-ferroprotoporphyrin complex. 3. Activation of latent enzyme may be accomplished with sulfhydryl compounds or photochemically, as well as with ascorbate. The nature of the action spectrum and the inhibition of activation by catalase implicates photochemically generated hydrogen peroxide as the chemical mediator of the photoactivation. 4. Spectral studies indicate that reductive or photochemical activation of the enzyme is not due to net reduction of the ferriprotoporphyrin, but rather to the reduction of an unidentified enzyme component which then permits the ferriprotoporphyrin prosthetic group to undergo reduction by the substrate, tryptophan. 5. p-Chloromercuribenzoate competitively inhibits tryptophan oxygenase, indicating probable involvement of one or more sulfhydryl groups as binding sites for tryptophan.