Active site rearrangement of the 2-hydrazinopyridine adduct in Escherichia coli amine oxidase to an azo copper(II) chelate form: a key role for tyrosine 369 in controlling the mobility of the TPQ-2HP adduct

Adduct I (lambda(max) at approximately 430 nm) formed in the reaction of 2-hydrazinopyridine (2HP) and the TPQ cofactor of wild-type Escherichia coli copper amine oxidase (WT-ECAO) is stable at neutral pH, 25 degrees C, but slowly converts to another spectroscopically distinct species with a lambda(max) at approximately 530 nm (adduct II) at pH 9.1. The conversion was accelerated either by incubation of the reaction mixture at 60 degrees C or by increasing the pH (>13). The active site base mutant forms of ECAO (D383N and D383E) showed spectral changes similar to WT when incubated at 60 degrees C. By contrast, in the Y369F mutant adduct I was not stable at pH 7, 25 degrees C, and gradually converted to adduct II, and this rate of conversion was faster at pH 9. To identify the nature of adduct II, we have studied the effects of pH and divalent cations on the UV-vis and resonance Raman spectroscopic properties of the model compound of adduct I (2). Strikingly, it was found that addition of Cu2+ to 2 at pH 7 gave a product (3) that exhibited almost identical spectroscopic signatures to adduct II. The X-ray crystal structure of 3 shows that it is the copper-coordinated form of 2, where the +2 charge of copper is neutralized by a double deprotonation of 2. These results led to the proposal that adduct II in the enzyme is TPQ-2HP that has migrated onto the active site Cu2+. The X-ray crystal structure of Y369F adduct II confirmed this assignment. Resonance Raman and EPR spectroscopy showed that adduct II in WT-ECAO is identical to that seen in Y369F. This study clearly demonstrates that the hydrogen-bonding interaction between O4 of TPQ and the conserved Tyr (Y369) is important in controlling the position and orientation of TPQ in the catalytic cycle, including optimal orientation for reactivity with substrate amines.     

Heterogeneity of the glucocorticoid receptors: molecular transformations during activation, detected by electrofocusing

Isoelectric focusing (IEF) of glucocorticoid receptor (GR) of the neural retina of the 14-day chick embryo was conducted under conditions that yielded quantitative recovery of binding activity. IEF of the cytosol, equilibrated with [3H]triamcinolone acetonide (TA) at 0-2 degrees C yielded three major TA-GR components with apparent isoelectric points (pI') of 5.4 +/- 0.3, 6.5 +/- 0.2, and 7.6 +/- 0.3, designated as I, II, and III, respectively. During temperature-induced activation (incubation at 30 degrees C for 60 min, in the presence of free [3H]TA and 0.15 M KCl), approximately 25% of the specifically bound TA was irreversibly lost. IEF reveals that this loss is accounted for by the complete loss of binding from I. During activation, II also decreases but correspondingly III increases, i.e., the sum of II and III remains unchanged. Only the bound TA of I is sensitive to the addition of KCl (a promoter of activation). This sensitivity of I is temperature dependent. Molybdate (an inhibitor of activation) protects the bound TA of I and suppresses the formation of III. These two effects of molybdate diminish simultaneously when the temperature is increased to 30 degrees C. III preferentially exhibits binding activity to nuclei. The data suggest that (i) the glucocorticoid-free cytosol contains two GRs, I and II, with possibly two different functions; (ii) activation involves the loss of bound TA from I and the transformation of II to III with increased pI; (iii) these two molecular events in GR activation are interdependent.     

Mediation by indole analogues of electron transfer during oxygen activation in variants of Escherichia coli ribonucleotide reductase R2 lacking the electron-shuttling tryptophan 48

Activation of dioxygen by the carboxylate-bridged diiron(II) cluster in the R2 subunit of class I ribonucleotide reductase from Escherichia coli results in the one-electron oxidation of tyrosine 122 (Y122) to a stable radical (Y122*). A key step in this reaction is the rapid transfer of a single electron from a near-surface residue, tryptophan 48 (W48), to an adduct between O(2) and diiron(II) cluster to generate a readily reducible cation radical (W48(+)(*)) and the formally Fe(IV)Fe(III) intermediate known as cluster X. Previous work showed that this electron injection step is blocked in the R2 variant with W48 replaced by phenylalanine [Krebs, C., Chen, S., Baldwin, J., Ley, B. A., Patel, U., Edmondson, D. E., Huynh, B. H., and Bollinger, J. M., Jr. (2000) J. Am. Chem. Soc. 122, 12207-12219]. In this study, we show that substitution of W48 with alanine similarly disables the electron transfer (ET) but also permits its chemical mediation by indole compounds. In the presence of an indole mediator, O(2) activation in the R2-W48A variant produces approximately 1 equiv of stable Y122* and more than 1 equiv of the normal (micro-oxo)diiron(III) product. In the absence of a mediator, the variant protein generates primarily altered Fe(III) products and only one-fourth as much stable Y122* because, as previously reported for R2-W48F, most of the Y122* that is produced decays as a consequence of the inability of the protein to mediate reductive quenching of one of the two oxidizing equivalents of the initial diiron(II)-O(2) complex. Mediation of ET is effective in W48A variants containing additional substitutions that also impact the reaction mechanism or outcome. In the reaction of R2-W48A/F208Y, the presence of mediator suppresses formation of the Y208-derived diiron(III)-catecholate product (which is predominant in R2-F208Y in the absence of reductants) in favor of Y122*. In the reaction of R2-W48A/D84E, the presence of mediator affects the outcome of decay of the peroxodiiron(III) intermediate known to accumulate in D84E variants, increasing the yield of Y122* by as much as 2.2-fold to a final value of 0.75 equiv and suppressing formation of a 490 nm absorbing product that results from decay of the two-electron oxidized intermediate in the absence of a functional ET apparatus.     

Direct photo-induced DNA strand scission by a ruthenium bipyridyl complex

Irradiation of plasmid DNA in the presence of Ru(II)-2, a modified tris(2,2'-bipyridyl)Ru(II) complex, in which two hydroxamic acid groups are attached to one of the three bipyridyl ligands, results in total fragmentation of the DNA. The photo-chemical reaction products were analyzed by gel electrophoresis, which revealed complete fragmentation. Further evidence for the complete degradation of the DNA was obtained by imaging the pre- and post-treated plasmid DNA using atomic force microscopy (AFM). A mechanism for the reaction is proposed. It initially involves the photo-chemical generation of Ru(III) ions and superoxide radicals, as corroborated by absorbance difference spectroscopy and electron paramagnetic resonance (EPR). Consequently, Ru(III) preferentially oxidizes guanine, liberating superoxide radicals that yield OH radicals. The OH radicals were identified by observing the spectral change at 532 nm of a 5'-dAdG substrate forming a colored adduct with thiobarbituric acid. These radicals are associated with the major non-specific damage exerted to DNA.     

Superoxide modulates the activity of myeloperoxidase and optimizes the production of hypochlorous acid.

Myeloperoxidase catalyses the conversion of H2O2 and Cl- to hypochlorous acid (HOCl). It also reacts with O2- to form the oxy adduct (compound III). To determine how O2- affects the formation of HOCl, chlorination of monochlorodimedon by myeloperoxidase was investigated using xanthine oxidase and hypoxanthine as a source of O2- and H2O2. Myeloperoxidase was mostly converted to compound III, and H2O2 was essential for chlorination. At pH 5.4, superoxide dismutase (SOD) enhanced chlorination and prevented formation of compound III. However, at pH 7.8, SOD inhibited chlorination and promoted formation of the ferrous peroxide adduct (compound II) instead of compound III. We present spectral evidence for a direct reaction between compound III and H2O2 to form compound II, and for the reduction of compound II by O2- to regenerate native myeloperoxidase. These reactions enable compound III and compound II to participate in the chlorination reaction. Myeloperoxidase catalytically inhibited O2- -dependent reduction of Nitro Blue Tetrazolium. This inhibition is explained by myeloperoxidase undergoing a cycle of reactions with O2-, H2O2 and O2-, with compounds III and II as intermediates, i.e., by myeloperoxidase acting as a combined SOD/catalase enzyme. By preventing the accumulation of inactive compound II, O2- enhances the activity of myeloperoxidase. We propose that, under physiological conditions, this optimizes the production of HOCl and may potentiate oxidant damage by stimulated neutrophils.     

Ferrylmyoglobin-catalyzed linoleic acid peroxidation

The addition of linoleic acid (18:2) to a solution containing oxymyoglobin (MbIIO2), metmyoglobin (MbIII), or metmyoglobin-azide complex (MbIII-N3-) resulted in the formation of a common complex with identical absorption spectral properties. The addition of H2O2 to a MbIII/linoleic acid mixture revealed a spectral profile with lambda max at 530 nm and different from that observed in the reaction of MbIII with H2O2 and identical to that of ferrylmyoglobin. This was accompanied by a progressive decrease in the absorption in the visible region, indicating heme degradation during the lipid peroxidation process. The oxidation products of linoleic acid during the MbIII/18:2/H2O2 interaction were assessed by HPLC under anaerobic and aerobic conditions. In both instances, the chromatograms at lambda 234 nm revealed the formation of a main peak with a retention time of 11.1 min, which cochromatographed with a standard of 9-hydroperoxide of linoleic acid. The latter adduct was not degraded by the oxoferryl complex of myoglobin. The conclusions originating from this research are two-fold. On the one hand, the identical spectral properties exhibited by the product originating from the reaction of either MbIIO2 or MbIII with linoleic acid bridge the apparent discrepancy between the different reactivities of MbIIO2 and MbIII toward H2O2 and their ability to promote lipid peroxidation. On the other hand, the pattern of oxidation products of linoleic acid observed during the MbIII/H2O2 interaction, i.e., the formation of a 9-hydroperoxide adduct as a major product, points to a specific binding character and a regioselectivity of the oxoferryl complex in the oxidation of unsaturated fatty acids or a catalytic preference for decomposition of the various isomeric hydroperoxides over that of the 9-hydroperoxide.     

Reaction of Co(II)bleomycin with dioxygen.

The reaction of Co(II)bleomycin with dioxygen has been investigated. Dioxygen binds to the Co(II) complex within the time of mixing according to electron spin resonance and uv-visible spectroscopy and dioxygen analysis. Then, two dioxygenated cobalt centers react, releasing 1 mol of O2 and forming an intermediate characterized by a few highly shifted 1H NMR resonances and loss of the ESR spectrum. This is thought to be a dioxygen-bridged dimer of cobalt bleomycin molecules. Time-dependent absorbance and dioxygen measurements yield the same second order rate constant for this step of the reaction. According to uv-visible and NMR spectral analysis, the intermediate decays into diamagnetic products in a first order rate process. High performance liquid chromatography and 1H NMR studies demonstrate that the product contains two bleomycin species of equal concentration. One component is Co(III)bleomycin, designated Form II. The other is the peroxide adduct of Co(III)bleomycin, Form I, as determined by direct determination of hydrogen peroxide, which is slowly released from the product at low pH. In contrast, hydrogen peroxide is readily detected during the reaction of Co(II)Blm with O2. In isolation, Form I is unstable at pH 7 and is converted within 24 h into a mixture of Form I and Form II.     

Reactivity of 2'-deoxyoxanosine, a novel DNA lesion.

2'-Deoxyoxanosine (dOxo) is a novel DNA lesion produced from 2'-deoxyguanosine by the reaction with nitrous acid or nitric oxide. We found that dOxo reacted with glycine under physiological conditions. The product was identified by spectrometric data as an adduct between the six membered ring of dOxo and an amino group of glycine. The adduct was more stable than dOxo under physiological conditions. The incubation of an oligodeoxynucleotide containing dOxo with glycine gave also rise to the adduct. These results suggest that dOxo formed in DNA reacts with amino groups of various compounds around DNA in vivo resulting in the adduct.     

Oxygenated iron bleomycin. A short-lived intermediate in the reaction of ferrous bleomycin with O2.

The reaction of Fe(II) . bleomycin with O2 to yield Fe(III) . bleomycin has been resolved into two kinetic events by stopped-flow spectrophotometry. The first event is first order with respect to both bleomycin and O2 and may be regarded as a second order reaction (k = 6.1 x 10(3) M-1s-1 at 2 degrees C). The first product has no EPR spectrum. The optical spectrum resembles those of Fe(II) . bleomycin complexes with CO, NO, and ethyl isocyanide. We propose that the first product is an Fe(II) . bleomycin . O2 complex. The second kinetic event is first order with respect to the first accumulated product (k = 0.11 s-1 at 2 degrees C) and independent of oxygen concentration. The product of this reaction is indistinguishable from Fe(III) . bleomycin by optical and EPR spectroscopy.     

Catalytic oxidation of p-cresol by ascorbate peroxidase

Transient and steady state kinetics, together with a range of chromatographic and spectroscopic techniques, have been used to establish the mechanism and the products of the H(2)O(2)-dependent oxidation of p-cresol by ascorbate peroxidase (APX). HPLC, GC-MS, and NMR analyses are consistent with the formation of 2, 2'-dihydroxy-5,5'-dimethylbiphenyl (II) and 4alpha,9beta-dihydro-8, 9beta-dimethyl-3(4H)-dibenzofuranone (Pummerer's ketone, III) as the major products of the reaction. In the presence of cumene hydroperoxide, two additional products were observed which, from GC and MS analyses, were shown to be 1,1-dimethylbenzylalcohol (IV) and bis-(1-methyl-1-phenyl-ethyl)-peroxide (V). The product ratio II:III was dependent on enzyme concentration: at low concentrations Pummerer's ketone (III) predominates and at high concentrations formation of the biphenyl compound (II) is favored. Steady-state data showed a sigmoidal dependence on [p-cresol] that was consistent with the presence of 2.01 +/- 0.15 binding sites for the substrate (25.0 degrees C, sodium phosphate, pH 7.0, mu = 2.2 mM) and independent of ionic strength in the range 2.2-500 mM. Single turnover kinetic experiments (pH 7.0, 5.0 degrees C, mu = 0.10 M) yielded second-order rate constants for Compound I reduction by p-cresol, k(2), of 5.42 +/- 0.10 x 10(5) M(-1) s(-1), respectively. Rate-limiting reduction of Compound II by p-cresol, k(3), showed saturation kinetics, giving values for K(d) = 1.54 +/- 0.12 x 10(-3) M and k(3) = 18.5 +/- 0.7 s(-1). The results are discussed in the more general context of APX-catalyzed aromatic oxidations.     

Interaction of tert-butyl hydroperoxide with hypochlorous acid. A spin trapping and chemiluminescence study

The formation of radical species during the reaction of ter-tbutyl hydroperoxide and hypochlorous acid has been investigated by spin trapping and chemiluminescence. A superposition of two signals appeared incubating tert-butyl hydroperoxide with hypochlorous acid in the presence of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN). The first signal (aN = 1.537 mT, aH beta = 0.148 mT) was an oxidation product of POBN caused by the action of hypochlorous acid. The second spin adduct (aN = 1.484 mT, aH beta = 0.233 mT) was derived from a radical species that was formed in the result of reaction of tert-butyl hydroperoxide with hypochlorous acid. Similarly, a superposition of two signals was also obtained using the spin trap N-tert-butyl-alpha-phenylnitrone (PBN). tert-Butyl hydroperoxide was also treated with Fe2+ or Ce4+ in the presence of POBN. Using Fe2+ a spin adduct with a N = 1.633 mT and aH beta = 0.276 mT was observed. The major spin adduct formed with Ce4+ was characterised by a N = 1.480 mT and aH beta = 0.233 mT. The reaction of tert-butyl hydroperoxide with hypochlorous acid was accompanied by a light emission, that time profile and intensity were identical to those emission using Ce4+. The addition of Fe2+ to tert-butyl hydroperoxide yielded a much smaller chemiluminescence. Thus, tert-butyl hydroperoxide yielded in its reaction with hypochlorous acid or Ce4+ the same spin adduct and the same luminescence profile. Because Ce4+ is known to oxidize organic hydroperoxides to peroxyl radical species, it can be concluded that a similar reaction takes place in the case of hypochlorous acid.     

Free radical intermediates formed during the oxidation of cyanide by horseradish peroxidase/H2O2 as detected with nitroso spin traps

Aqueous solutions of cyanide react with hydrogen peroxide/horseradish peroxidase and form the cyanyl radical, which can be trapped by 2-methyl-2-nitrosopropane (t-nitrosobutane, tNB) at pH 9.8. At lower pH a variety of radical adducts are formed; at higher pH, the main product was the spin adduct of the formamide radical with tNB. The use of deuterated tNB and 15N-labeled potassium cyanide allowed the observation of the very small nitrogen coupling of this radical adduct. Experiments using 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS) as the spin trap yielded only the formamide radical adduct, which was identified by an independent synthesis starting from formamide. Both hydrogen splittings of its amino group could be resolved using deuterated DBNBS as the spin trap.     

Oxygen activation by a mixed-valent, diiron(II/III) cluster in the glycol cleavage reaction catalyzed by myo-inositol oxygenase

myo-Inositol oxygenase (MIOX) catalyzes the ring-cleaving, four-electron oxidation of its cyclohexan-(1,2,3,4,5,6-hexa)-ol substrate (myo-inositol, MI) to d-glucuronate (DG). The preceding paper [Xing, G., Hoffart, L. M., Diao, Y., Prabhu, K. S., Arner, R. J., Reddy, C. C., Krebs, C., and Bollinger, J. M., Jr. (2006) Biochemistry 45, 5393-5401] demonstrates by M?ssbauer and electron paramagnetic resonance (EPR) spectroscopies that MIOX can contain a non-heme dinuclear iron cluster, which, in its mixed-valent (II/III) and fully oxidized (III/III) states, is perturbed by binding of MI in a manner consistent with direct coordination. In the study presented here, the redox form of the enzyme that activates O(2) has been identified. l-Cysteine, which was previously reported to accelerate turnover, reduces the fully oxidized enzyme to the mixed-valent form, and O(2), the cosubstrate, oxidizes the fully reduced form to the mixed-valent form with a stoichiometry of one per O(2). Both observations implicate the mixed-valent, diiron(II/III) form of the enzyme as the active state. Stopped-flow absorption and freeze-quench EPR data from the reaction of the substrate complex of mixed-valent MIOX [MIOX(II/III).MI] with limiting O(2) in the presence of excess, saturating MI reveal the following cycle: (1) MIOX(II/III).MI reacts rapidly with O(2) to generate an intermediate (H) with a rhombic, g < 2 EPR spectrum; (2) a form of the enzyme with the same absorption features as MIOX(II/III) develops as H decays, suggesting that turnover has occurred; and (3) the starting MIOX(II/III).MI complex is then quantitatively regenerated. This cycle is fast enough to account for the catalytic rate. The DG/O(2) stoichiometry in the reaction, 0.8 +/- 0.1, is similar to the theoretical value of 1, whereas significantly less product is formed in the corresponding reaction of the fully reduced enzyme with limiting O(2). The DG/O(2) yield in the latter reaction decreases as the enzyme concentration is increased, consistent with the hypothesis that initial conversion of the reduced enzyme to the MIOX(II/III).MI complex and subsequent turnover by the mixed-valent form is responsible for the product in this case. The use of the mixed-valent, diiron(II/III) cluster by MIOX represents a significant departure from the mechanisms of other known diiron oxygenases, which all involve activation of O(2) from the II/II manifold.     

Pyrolytic behavior of waste corn cob

The powder of the agricultural waste corn cob was pyrolyzed in a tube-typed stainless steel reactor of 200 ml volume under N2 atmosphere. The compositions of the gases and liquid obtained at different pyrolytic temperatures below 600 degrees C at the heating rate of 30 K/min were analyzed. With the increment of the pyrolytic temperature, the yields of the solid and the liquid products were decreased, but the yield of gas products was increased. The liquid products were approximately 34-40.96% (wt%), the gas products were 27-40.96% (wt%) and the solid products 23.6-31.6% (wt%). There were less changes for the yields of these products above 600 degrees C. The gas products were analyzed by gas chromatography (GC) as CO2, CO, H2, CH4, C2H4, C3H6, C3H8, etc. When the temperature was 350-400 degrees C, the gases had CO2 and CO 80-95% (v/v). When the temperature increased continuously, yields of H2, CH4, C2H4, C3H6 and C3H8 gradually increased. The liquid products were identified by GC-MS as phenols, 2-furanmethanol, 2-cyclopentanedione, etc. The Fourier transform infra-red spectrophotometer (FT-IR) analysis of the liquid product showed a strong -OH group absorption peak. Differential thermogravimetric analysis (DTG) showed that thermal decomposition process involves two steps. The heating rate affects not only the activation energy of the decomposition reaction, but also the path of the reaction. With the increment of the heating rate, the maximum rate temperature of the decomposition reaction was shifted to a higher temperature, and the order and activation energy of the total decomposition reaction were decreasing.     

Addition of oxygen to the diiron(II/II) cluster is the slowest step in formation of the tyrosyl radical in the W103Y variant of ribonucleotide reductase protein R2 from mouse

Activation of O2 by the diiron(II/II) cluster in protein R2 of class I ribonucleotide reductase generates the enzyme's essential tyrosyl radical. A crucial step in this reaction is the transfer of an electron from solution to a diiron(II/II)-O2 adduct during formation of the radical-generating, diiron(III/IV) intermediate X. In the reaction of R2 from Escherichia coli, this electron injection is initiated by the rapid (>400 s-1 at 5 degrees C), transient oxidation of the near-surface residue, tryptophan 48, to a cation radical and is blocked by substitution of W48 with F, A, G, Y, L, or Q. By contrast, a study of the cognate reaction in protein R2 from mouse suggested that electron injection might be the slowest step in generation of its tyrosyl radical, Y177* [Schmidt, P. P., Rova, U., Katterle, B., Thelander, L., and Gr?slund, A. (1998) J. Biol. Chem. 273, 21463-21472]. The crucial evidence was the observation that Y177* production is slowed by approximately 30-fold upon substitution of W103, the cognate of the electron-shuttling W48 in E. coli R2, with tyrosine. In this work, we have applied stopped-flow absorption and freeze-quench electron paramagnetic resonance and M?ssbauer spectroscopies to the mouse R2 reaction to evaluate the possibility that an already sluggish electron-transfer step is slowed by 30-fold by substitution of this key residue. The drastically reduced accumulation of cluster X, failure of precursors to the intermediate to accumulate, and, most importantly, first-order dependence of the rate of Y177* formation on the concentration of O2 prove that addition of O2 to the diiron(II/II) cluster, rather than electron injection, is the slowest step in the R2-W103Y reaction. This finding indicates that the basis for the slowing of Y177* formation by the W103Y substitution is an unexpected secondary effect on the structure or dynamics of the protein, its diiron(II/II) cluster, or both rather than the expected chemical effect on the electron injection step.     

Enzyme-catalyzed and enzyme-triggered pathways in dioxygenation of 1-monolinoleoyl-rac-glycerol by potato tuber lipoxygenase

It was shown for the first time that potato tuber lipoxygenase (ptLOX) catalyzed the aerobic oxidation of 1-monolinoleoyl-rac-glycerol (mLG) in a mixed micellar reaction solution with the non-ionic detergent monododecyl ether of decaoxyethylene glycol. No hydrolysis of mLG occurred during the reaction. The four major reaction products obtained at 23 degrees C were identified as 1-[9-hydroperoxy-10E,12Z-octadecadienoyl]-rac-glycerol (9-(E,Z)HPODE-GE, 41%), 1-[13-hydroperoxy-9Z,11E-octadecadienoyl]-rac-glycerol (13-(Z,E)-HPODE-GE, 17%), and their all-trans isomers ( approximately 21% each). The molar fraction of all-trans isomers depended on the temperature of the reaction solution; it was found that at 0 degrees C their molar fractions were approximately 15.5% each, while 9-(E,Z)HPODE-GE and 13-(Z,E)-HPODE-GE gave 42% and 27%, respectively, of the overall product. A free radical scavenger, 4-hydroxy-TEMPO, dramatically increased the molar fraction of 9-(E,Z)HPODE-GE, yielding 83% at 23 degrees C, at the expense of all other products. Chiral HPLC of 9-(E,Z)HPODE-GE formed in the presence of 4-hydroxy-TEMPO revealed that it was composed of approximately 94% S and approximately 6% (R) isomers. This assures largely a uniform orientation of mLG molecules in the ptLOX active center, with their methyl end most likely deepened into the protein globule. The second major product, 13-(Z,E)-HPODE-GE, which yielded approximately 9% of the total product formed in the presence of 4-hydroxy-TEMPO, was racemic, and so were the all-trans isomers. Therefore, the last three cannot be considered the true products of the enzyme reaction, which is known to be stereospecific. It appears that they were formed as a result of (i) leakage of the pentadienyl radicals from the ptLOX active center and their subsequent non-enzymatic dioxygenation, and/or (ii) leakage of the peroxyl radicals leading to a free radical chain reaction affording all positional, geometrical and stereoisomers of the products. This reaction resembles ptLOX oxidation of another non-ionizable substrate, linoleyl alcohol [I.A. Butovich, S.M. Luk'yanova, C.C. Reddy, Arch. Biochem. Biophys. 378 (2000) 65-77], and differed substantially from oxidation of ionizable linoleic acid. Consequently, formation of large amounts of the non-specific oxidation products might be considered a universal characteristic of ptLOX oxidation of non-ionizable compounds.     

Heme-copper/dioxygen adduct formation relevant to cytochrome c oxidase: spectroscopic characterization of [(6L)FeIII-(O2 2−)-CuII]+

In the further development and understanding of heme-copper dioxygen reactivity relevant to cytochrome c oxidase O(2)-reduction chemistry, we describe a high-spin, five-coordinate dioxygen (peroxo) adduct of an iron(II)-copper(I) complex, [((6)L)Fe(II)Cu(I)](BArF(20)) (1), where (6)L is a tetraarylporphyrinate with a tethered tris(2-pyridylmethyl)amine chelate for copper. Reaction of 1 with O(2) in MeCN affords a remarkably stable [t(1/2) (rt; MeCN) approximately 60 min] adduct, [((6)L)Fe(III)-(O(2) (2-))-Cu(II)](+) (2) [EPR silent; lambda(max)=418 (Soret), 561 nm], formulated as a peroxo complex based on manometry (1:O(2)=1:1; spectrophotometric titration, -40 degrees C, MeCN), mass spectrometry {MALDI-TOF-MS: (16)O(2), m/z 1191 ([((6)L)Fe(III)-((16)O(2) (2-))-Cu(II)](+)); (18)O(2), m/z 1195}, and resonance Raman spectroscopy (nu((O-O))=788 cm(-1); Delta(16)O(2)/(18)O(2)=44 cm(-1); Delta(16)O(2)/(16/18)O(2)=22 cm(-1)). (1)H and (2)H NMR spectroscopy (-40 degrees C, MeCN) reveals that 2 is the first heme-copper peroxo complex which is high-spin, with downfield-shifted pyrrole resonances (delta(pyrrole)=75 ppm, s, br) and upfield shifted peaks at delta= -22, -35, and -40 ppm, similar to the pattern observed for the mu-oxo complex [((6)L)Fe(III)-O-Cu(II)](BAr(F)) (3) (known S=2 system, antiferromagnetically coupled high-spin Fe(III) and Cu(II)). The corresponding magnetic moment measurement (Evans method, CD(3)CN, -40 degrees C) also confirms the S=2 spin state, with mu(B)=4.9. Structural insights were obtained from X-ray absorption spectroscopy, showing Fe-O (1.83 A) and Cu-O (1.882 A) bonds, and an Fe...Cu distance of 3.35(2) A, suggestive of a mu-1,2-peroxo ligand present in 2. The reaction of 2 with cobaltocene gives 3, differing from the observed full reduction seen with other heme-Cu peroxo complexes. Finally, thermal decomposition of 2 yields 3, with concomitant release of 0.5 mol O(2) per mol 2, as confirmed quantitatively by an alkaline pyrogallol dioxygen scavenging solution.     

Identification of form III conformers in tRNAPhe from Escherichia coli by intramolecular photo-cross-linking

In the absence of divalent cations, at neutral pH, low ionic strength, and low to moderate temperature, tRNAs are known to be in a denatured form, designated form III in the tRNA phase diagram by Cole et al. [Cole, P. E., Yang, S. R., & Crothers, D. M. (1972) Biochemistry 11, 4358-4368]. Form III tRNAPhe from Escherichia coli has been studied at pH 7, 5 mM Na+, and 10 degrees C. As judged from ethidium bromide intercalation, it exhibits extensive secondary structure. 4-Thiouridine in position 8 of the tRNAPhe sequence was used as a built-in photoaffinity probe. Spectroscopic and spectrofluorometric analysis in the near-UV range of form III tRNAPhe irradiated with broad-band near-UV light to completion of the reaction before or after reduction with NaBH4 revealed that the Pdo(4-5)Cyt (8-C) and Pdo(4-5)Urd (8-U) adducts form in equimolar yield. In different experiments, the overall yield of s4U conversion to these adducts varies between 20 and 40%. The remaining s4U is photolyzed to weakly absorbing product(s) in the near-UV range. The disappearance of s4U follows biexponential kinetics while the 8-C adduct formation follows monoexponential kinetics, indicating the presence of at least two tRNA classes of conformers, not in equilibrium on the time scale of the reaction. Migration on a denaturing polyacrylamide gel of irradiated form III tRNAPhe revealed three main bands, D1, D2, and D3, and no slowly migrating tRNA dimers. D1 migrates at the control position and presumably contains the photolysis product(s) P. The fast-migrating D2 and D3 bands contain 8-Pyr cross-links which were identified by sequence analysis as 8-(66-68) in D2 and 8-(40-43) and 8-(59-62) in D3. On the basis of these data, it is proposed that the minor poorly photoreactive class II conformers are the cloverleaf and close variants, whereas the major class I cross-linkable conformers are essentially long-extended secondary structures. Clearly, our data demonstrate the polymorphism of form III tRNAPhe.     

Reactions of the carcinogen N-acetoxy-4-acetamidostilbene with nucleosides.

The carcinogen N-acetoxy-4-acetamidostilbene (N-AcO-AAS) yields multiple products in reactions with guanosine, adenosine or cytidine in aqueous acetone. The major product from the reaction with cytidine is a deamination product, 1-(4-acetamidophenyl)-1-(3-uridyl)-2-hydrosy-2-phenylethane. Three minor products were unstable and were characterized only by their UV spectra and pK values. Adenosine yielded two major products, one of them 1-(4-acetamidophenyl)-1-(N6-adenoxyl)-2-hydroxy-2-phenylethane, and the second 3-(beta-D-ribosyl)-7-phenyl-8-(4-acetamidophenyl)-7,8 dihydroimidazo [2,1-i] purine. The major adduct with guanosine is 1-(4-acetamidophenyl)-1-(1-guanosyl)-2-hydroxy-2-phenylethane. One minor adduct also appears to be a guanosine-N-1 derivative, while two other minor adducts yield 1-(4-acetamidophenyl)-2-phenyl-1, 2-ethanediol on acid hydrolysis, and thus appear to be O6-derivatives. None of the guanine adducts isolated had the properties of N-7, C-8 or N2 adducts. In this respect, N-Aco-AAS appears to behave more like a classical alkylating agent than like previously studied N-acetoxy-N-arylacetamides, although the target organs of 4-acetamidostilbene are the same as those of other N-arylacetamides.     

New substrates for the radioassay of angiotensin converting enzyme of endothelial cells in culture.

To develop means of measuring angiotensin converting enzyme of endothelial cells in culture, we have synthesized benzoyl-Phe-Ala-Pro-OH (I), benzoyl-Pro-Phe-Arg-OH (II) and benzoyl-Gly-His-Leu-OH (III), each bearing a 3H-atom on the para-position of its benzoyl moiety. All three of the acylated tripeptides are substrates for the enzyme. Substrate I exhibits the lowest Km (12.5 micrometer) and yields the most sensitive assay: the enzyme of 10(6) cells can be measured in a 30 min incubation at 37 degrees C. Radiolabelled reaction product is separated from substrate by extraction of acidified reaction mixture with an organic solvent, and the rate of formation of product can be quantified by liquid scintillation counting of the organic phase. Substrate III can also be used to measure angiotensin converting enzyme of cells but requires longer incubations (180--240 min) and high salt concentrations (0.75 M Na2SO4). Substrate II is not specific: it is hydrolyzed by more than one enzyme of endothelial cells.