Reductive addition of glutathione to p-benzoquinone, 2-hydroxy-p-benzoquinone, and p-benzoquinone epoxides. Effect of the hydroxy- and glutathionyl substituents on p-benzohydroquinone autoxidation

The reductive addition of GSH to p-benzoquinones, 2-hydroxy-p-benzoquinone, and 2,3-epoxy-p-benzoquinones with different degree of methyl substitution was studied in terms of absorption spectral changes and autoxidation reactions. The nucleophilic addition of GSH to p-benzoquinone yields a glutathionyl-p-benzohydroquinone product with maximal absorption at lambda 303nm. This compound autoxidizes slowly--but at a rate 8-fold higher than the parent hydroquinone--to glutathionyl-p-benzoquinone, which reveals maximal absorption at lambda 367 nm. The autoxidation of the glutathionyl derivative is accompanied by O2 consumption and H2O2 formation. The nucleophilic addition of GSH to either 2-hydroxy-p-benzoquinone or 2,3-epoxy-p-benzoquinone yields the same primary molecular product, 2-hydroxy-5-glutathionyl-p-benzohydroquinone, a compound that shows maximal absorption at lambda 300 nm and autoxidizes at rates substantially higher (44-fold) than the parent glutathionyl hydroquinone lacking a -OH substituent. The autoxidation product, 2-hydroxy-5-glutathionyl-p-benzoquinone, reveals maximal absorbance at lambda 343 nm as well as a resolved absorption band at longer wavelengths (lambda 520 nm), the latter contributed by the -OH substituent. The glutathionyl substituent exerted only minor changes in the reduction potential of the quinones, whereas the -OH substituent lowered significantly the half-wave reduction potential, as measured in aqueous solutions. The rate of autoxidation was markedly enhanced by both substituents as follows: hydroxy-glutathionyl-p-benzohydroquinone much greater than hydroxy-p-benzohydroquinone much greater than glutathionyl-p-benzohydroquinone greater than p-benzohydroquinone. Superoxide dismutase enhanced the rate of autoxidation of p-benzohydroquinone and its glutathionyl adduct, whereas it inhibited autoxidation of the hydroxy derivatives with or without glutathionyl substitution. The biochemical significance of these results is discussed in terms of the pro-oxidant character of the reductive addition of GSH to p-benzoquinones, alpha-hydroxyquinones, and quinone epoxides.     

DT-diaphorase-catalyzed two-electron reduction of quinone epoxides

DT-diaphorase catalyzes the two-electron reduction of the unsubstituted quinone epoxide, 2,3-epoxy-p-benzoquinone, at expense of NAD(P)H with formation of 2-OH-p-benzohydroquinone as the reaction product. The further conversion reactions of 2-OH-p-benzohydroquinone are influenced by the presence of O2 in the medium. Under aerobic conditions, 2-OH-p-benzohydroquinone undergoes autoxidation--probably with formation of 2-OH-semiquinone intermediates--to 2-OH-p-benzoquinone. The latter product is rapidly reduced by DT-diaphorase and, thus, its accumulation can be only observed upon exhaustion of NADPH. Under anaerobic conditions, 2-OH-p-benzohydroquinone does not undergo autoxidation and its accumulation is stoichiometrically (1:1) related to the amount of NADPH oxidized and epoxide substrate reduced. DT-diaphorase also catalyzes the reduction of the disubstituted quinone epoxide, 2,3-dimethyl-2,3-epoxy-1,4-naphthoquinone. Neither the aliphatic epoxide, trans-stilbene oxide, nor the aromatic epoxide, 4,5-epoxy-benzo[a]pyrene are substrates for DT-diaphorase. The reduction of 2,3-epoxy-p-benzoquinone is also catalyzed by the one-electron transfer enzyme, NADPH-cytochrome P450 reductase at a rate similar to that found with DT-diaphorase. However, this reaction differs from that catalyzed by DT-diaphorase in the distribution of molecular products as well as in the relative contribution of nonenzymatic reactions, i.e. semiquinone disproportionation and autoxidation.     

1,4-Reductive addition of glutathione to quinone epoxides. Mechanistic studies with h.p.l.c. with electrochemical detection under anaerobic and aerobic conditions. Evaluation of chemical reactivity in terms of autoxidation reactions

The nucleophilic addition of GSH to quinonoid compounds, characterized as a 1,4-reductive addition of the Michael type, was studied with p-benzoquinone- and 1,4-naphthoquinone epoxides with different degree of methyl substitution. Identification and evaluation of molecular products from the above reaction were assessed by h.p.l.c. with either reductive or oxidative electrochemical detection, based on the redox properties retained in the molecular products formed. It was found that the degree of methyl substitution of the quinone epoxide, from either the 1,4-naphthoquinone- or p-benzoquinone epoxide series, determined their rate of reaction with GSH. The reductive addition implied the rearrangement of the quinone structure with opening of the epoxide ring yielding as the primary product a hydroxy-glutathionyl substituted adduct of either p-benzohydroquinone or 1,4-naphthohydroquinone. The primary product undergoes elimination reactions and redox transitions which bring about a number of secondary molecular products. The distribution pattern of the latter depends on the degree of methyl substitution of the quinone epoxide studied and on the concentration of O2 in the solution. The occurrence of the hydroxy-substituent in position alpha, adjacent to the carbonyl group, enhances the autoxidation properties of the compound resulting in an augmented O2 consumption and H2O2 production. Therefore, it could be expected that the chemical reactivity of the products originating from the thiol-mediated nucleophilic addition to quinone epoxides would be of toxicological interest.     

One-electron transfer reactions of diquat radical to different reduction intermediates of oxygen. Formation of hydroxyl radical and electronically excited states

The one-electron transfer activation of DQ++ by microsomal fractions comprises an aerobic phase and an anaerobic phase. The aerobic phase is characterized by O2 consumption, formation of electronically excited states with main emission below 600 nm, and H2O2 formation. The anaerobic phase is characterized by H2O2 consumption, DQ+ accumulation, HO. formation, and also electronically excited state formation with main emission beyond 600 nm. Superoxide dismutase abolishes the photoemission during the aerobic phase, whereas it has no effect on the photoemission originating during the anaerobic phase. The hydroxylation products of the aromatic compound salicylate, mainly 2,3- and 2,5-dihydroxybenzoic acids--indicative of the occurrence of HO.-, were detected by h.p.l.c. with oxidative electrochemical detection during the anaerobic phase, but not during the aerobic phase. Neither H2O2 consumption nor HO. are prevented by desferrioxamine. These experimental observations are interpreted on the grounds of two main electron-transfer reactions of DQ.+: under aerobic conditions, two one-electron transfer steps to molecular O2 and O2.- to yield H2O2. Under anaerobic conditions, one-electron transfer step to contaminating iron or any ferrioxamine formed to a ferrous complex which can support a Fenton-like reduction of H2O2 with formation of HO.. The toxicological relevance for the occurrence of such reactions is also discussed in terms of the formation of electronically excited states.     

Formation of electronically excited states during the interaction of p-benzoquinone with hydrogen peroxide

The reaction between p-benzoquinone and H₂O₂ in slightly alkaline solutions yields three major quinoid products that accumulate in the reaction mixture: (a) 2,3-epoxy-p-benzoquinone, (b) 2-hydroxy-p-benzoquinone and (c) p-benzohydroquinone. The reaction is accompanied by photoemission, probably originating from excited triplet 2-hydroxy-p-benzoquinone. These products originate from hydrogen peroxide and hydroxide nucleophilic addition to the C₂?C₃ double bond, as well as secondary redox interactions. The hydroxy substituent and the epoxide ring exert a substantial influence on the electronic distribution in the p-benzoquinone molecule leading to a decrease in the half-wave potential, as compared to the parent p-benzoquinone. The generation of electronically excited states is the result of reactions secondary to the nucleophilic additions involving 2-hydroxy-p-benzosemiquinone, H₂O₂ and hydroxyl radical. The process involves the primary oxidation of 2-hydroxy-p-benzosemiquinone by hydrogen peroxide, followed by oxidation of the semiquinone by hydroxyl radical leading to the formation of the electronically excited quinone. The decay of the excited triplet to the ground state is accompanied by photoemission with maximal intensity at 485–530 nm. Thermodynamic calculations along with an observed increase of photoemission intensity in anaerobiosis point to the triplet (n, π*) multiplicity of the excited state. The efficiency of chemiluminescence could be calculated as 10^?8 photons/2-hydroxy-p-benzoquinone molecule formed. Photoemission arising from the p-benzoquinone/H₂O₂ reaction was inhibited efficiently by addition of GSH to the reaction mixture. This may be due to deactivation of the triplet quinone by a 2-glutathionyl-p-benzohydroquinone adduct, involving thioether α-hydrogen atom-transfer to the triplet ketone.     

Reduction of ferrylmyoglobin to metmyoglobin by quinonoid compounds

Several quinoid compounds mediated the reduction of ferrylmyoglobin (MbIV) to metmyoglobin (MbIII). The efficiency of the MbIV reduction to MbIII was accomplished by the quinones in the following order: p-benzoquinone greater than 1,4-naphthoquinone greater than 2-OH-1,4-naphthoquinone greater than 2,3-epoxy-1,4-naphthoquinone. The quinone-mediated reduction of MbIV to MbIII had the following characteristics: (a) it was stoichiometrically--rather than catalytically--related to the number of cycles of the MbIV----MbIII transition involving the reduction of H2O2. (b) It proceeded with similar efficiencies under aerobic and anaerobic conditions. (c) It did not require the free radical form of MbIV(.MbIV), thus excluding a two-electron oxidation of the quinone. (d) the nucleophilic addition of--NH2 groups of the apoprotein on the quinone seemed not to be involved through an alternative pathway in the reduction of MbIV, especially since 2-OH-1,4-naphthoquinone, a compound which cannot undergo nucleophilic addition, also facilitated the reduction of the ferryl compound. (e) No two-electron oxidation products of the unsubstituted quinones, such as quinone epoxides, were detected in the spent reaction mixture analyzed by HPLC with electrochemical detection. On the basis of these observations, it is suggested that the reduction of MbIV to MbIII by the above quinonoid compounds is a one-electron transfer process, with electron abstraction being probably accomplished at some site in the benzo ring of the quinone.     

Oxidation of hydroquinone by both cellular and extracellular grapevine peroxidase fractions.

The oxidation of hydroquinone by two peroxidase (EC 1.11.1.7) fractions obtained from the cells and spent medium of cell cultures of grapevine (Vitis vinifera cv Monastrell) has been studied, and their comparative efficacy (kcat/KM ratio) studied in both the H2O2-consuming and hydroquinone-consuming reactions. While the efficacy in the H2O2-consuming reaction is practically identical for both enzyme fractions, the cellular peroxidase has five-fold more efficacy in the hydroquinone-consuming reaction than the peroxidase located in the spent medium. Screening of cellular peroxidases capable of oxidizing hydroquinone on polyacrylamide gels, by means of a staining reaction based on the nucleophilic attack of 4-aminoantipyrine on p-benzoquinone in acidic media, reveals that all the cellular peroxidase isoenzymes are capable of oxidizing hydroquinone, probably yielding a quinone-diimine as a product of the staining reaction. Since isoperoxidases found in cellular fractions are also present in the spent medium, the values found for the different efficacies in the hydroquinone-consuming reaction must be considered as the results of the different proportions in which each peroxidase isoenzyme was found in the two fractions. The localization of a benzoquinone-generating system of high efficacy inside the plant cell, and probably located in vacuoles, is discussed with respect to the harmful role which the quinone/semiquinone pair might play in cell death, as part of the hypersensitive response expressed within the mechanism of plant disease resistance.     

Enhancement of light emission in the bacterial luciferase reaction by H2O2

In the bacterial luciferase reaction, light emission is due to the mixed function oxidation of FMNH2 and long chain aldehydes, which leads to the formation of an electronically excited product species, postulated to be luciferase-bound 4a-hydroxy flavin. In the present work it was found that H2O2 stimulates an additional and kinetically distinct luminescence. The stimulation is more apparent in reactions inhibited by long chain alcohols, and the H2O2 is effective even if added secondarily. The stimulation requires H2O2 only at the outset; its subsequent destruction by catalase does not diminish the response, appreciably.     

On the mechanism of the Mn3(+)-induced neurotoxicity of dopamine:prevention of quinone-derived oxygen toxicity by DT diaphorase and superoxide dismutase

Dopamine (DA) is rapidly oxidized by Mn3(+)-pyrophosphate to its cyclized o-quinone (cDAoQ), a reaction which can be prevented by NADH, reduced glutathione (GSH) or ascorbic acid. The oxidation of DA by Mn3+, which appears to be irreversible, results in a decrease in the level of DA, but not in a formation of reactive oxygen species, since oxygen is neither consumed nor required in this reaction. The formation of cDAoQ can initiate the generation of superoxide radicals (O2-.) by reduction-oxidation cycling, i.e. one-electron reduction of the quinone by various NADH- or NADPH-dependent flavoproteins to the semiquinone (QH.), which is readily reoxidized by O2 with the concomitant formation of O2-.. This mechanism is believed to underly the cytotoxicity of many quinones. Two-electron reduction of cDAoQ to the hydroquinone can be catalyzed by the flavoprotein DT diaphorase (NAD(P)H:quinone oxidoreductase). This enzyme efficiently maintains DA quinone in its fully reduced state, although some reoxidation of the hydroquinone (QH2) is observed (QH2 + O2----QH. + O2-. + H+; QH. + O2----Q + O2-.). In the presence of Mn3+, generated from Mn2+ by O2-. (Mn2+ + 2H+ + O2-.----Mn3+ + H2O2) formed during the autoxidation of DA hydroquinone, the rate of autoxidation is increased dramatically as is the formation of H2O2. Furthermore, cDAoQ is no longer fully reduced and the steady-state ratio between the hydroquinone and the quinone is dependent on the amount of DT diaphorase present. The generation of Mn3+ is inhibited by superoxide dismutase (SOD), which catalyzes the disproportionation of O2-. to H2O2 and O2. It is noteworthy that addition of SOD does not only result in a decrease in the amount of H2O2 formed during the regeneration of Mn3+, but, in fact, prevents H2O2 formation. Furthermore, in the presence of this enzyme the consumption of O2 is low, as is the oxidation of NADH, due to autoxidation of the hydroquinone, and the cyclized DA o-quinone is found to be fully reduced. These observations can be explained by the newly-discovered role of SOD as a superoxide:semiquinone (QH.) oxidoreductase catalyzing the following reaction: O2-. + QH. + 2H+----QH2 + O2. Thus, the combination of DT diaphorase and SOD is an efficient system for maintaining cDAoQ in its fully reduced state, a prerequisite for detoxication of the quinone by conjugation with sulfate or glucuronic acid. In addition, only minute amounts of reactive oxygen species will be formed, i.e. by the generation of O2-., which through disproportionation to H2O2 and further reduction by ferrous ions can be converted to the hydroxyl radical (OH.). Absence or low levels of these enzymes may create an oxidative stress on the cell and thereby initiate events leading to cell death.     

The effects of nitric oxide on the oxidations of l-dopa and dopamine mediated by tyrosinase and peroxidase

The effects of nitric oxide (NO) on both tyrosinase/O(2)- and horseradish peroxidase/H(2)O(2)-mediated oxidations of dopamine and its o-dihydric phenol precursor l-dopa were compared with autoxidative processes and quantitatively assessed by oxidative and reductive electrochemical detection systems. In peroxidase/H(2)O(2)/NO-catalyzed reactions, significantly more substrate was oxidized than in the corresponding control incubations lacking NO. In tyrosinase/O(2)/NO-promoted reactions the total amounts of l-dopa and dopamine oxidized were significantly less than the amounts of the substrates oxidized by enzyme alone. These data indicate that the activity of the heme protein peroxidase was enhanced by NO, whereas tyrosinase, a copper-containing monoxygenase, was inhibited. The NO-mediated reduction of tyrosinase/O(2) activity may be attributed to the formation of an inhibitory copper.nitrosyl complex. An oxidized nitrodopamine derivative, considered to be either the quinone or semiquinone of 6-nitrosodopamine, was generated in peroxidase/H(2)O(2)/NO-mediated reactions with dopamine along with two oxidized melanin precursors, dopamine quinone and dopaminechrome. No corresponding nitroso compound was formed in reactions involving l-dopa or in any of the tyrosinase-mediated reactions. The formation of such a noncyclized nitrosodopamine represents an important alternative pathway in catecholamine metabolism, one that by-passes the formation of cytoprotective indole precursors of melanin. The results of this investigation suggest that cellular integrity and function can be adversely affected by NO-promoted oxidations of dopamine and other catechols, reactions that not only accelerate their conversion to reactive quinones but also form potentially cytotoxic noncyclized nitroso derivatives. Reduced levels of dopamine in the brain through NO-enhanced oxidation of the catecholamine will almost certainly be manifested by diminished levels of the dopamine-derived brain pigment neuromelanin.     

The ligninase of Phanerochaete chrysosporium generates cation radicals from methoxybenzenes

The hemoprotein ligninase of Phanerochaete chrysosporium catalyzes, in the presence of H2O2, a variety of seemingly different oxidations in lignin and lignin model compounds. Here we show that the enzyme also catalyzes the oxidation of various methoxybenzenes. ESR spectroscopy shows that the compounds are oxidized to aryl cation radicals. These decompose, evidently by H2O addition. Thus, 1,4-dimethoxybenzene is converted to p-benzoquinone and methanol. We propose a unified mechanism, based on formation of aryl cation radicals, to explain the various reactions catalyzed by the ligninase.     

Aromatic ring cleavage by homoprotocatechuate 2,3-dioxygenase: role of His200 in the kinetics of interconversion of reaction cycle intermediates

Homoprotocatechuate 2,3-dioxygenase (WT 2,3-HPCD) isolated from Brevibacterium fuscum utilizes an active site Fe(II) and O(2) to catalyze proximal extradiol cleavage of the aromatic ring of the substrate (HPCA). Here, the conserved active site residue His200 is changed to Gln, Glu, Ala, Asn, and Phe, and the reactions of the mutant enzymes are probed using steady-state and transient kinetic techniques. Each mutant catalyzes ring cleavage of HPCA to yield the normal product. H200Q and H200N retain 30-40% of the WT 2,3-HPCD activity at 24 degrees C, but the other mutants reduce the k(cat) to less than 9% of normal. The origin of the reduced activity is unlikely to be the substrate binding phase of the catalytic cycle, because the multistep anaerobic binding reaction of the chromophoric substrate 4-nitrocatechol (4NC) is shown to proceed with rate constants similar to those observed for WT 2,3-HPCD. In contrast, the rate constants of several steps in the multistep O(2) binding/insertion and product release half of the reaction cycle are substantially slowed, in particular the steps in which activated oxygen attacks the organic substrate and in which product is released. In the case of the H200N mutant, the product of 4NC oxidation is not the usual ring cleavage product, but rather the 4NC quinone. These results suggest that the main role of His200 is in facilitating the steps in the second half of the reaction cycle. The decreased rate constants for the O(2) insertion steps in the catalytic cycles of the mutant enzymes allow the oxygen adduct of an extradiol dioxygenase to be detected for the first time.     

Co-oxidation of salicylate and cholesterol during the oxidation of metmyoglobin by H2O2

The reaction between metmyoglobin and H2O2 proceeds with oxidation of the hemo-protein iron to a higher valence state and consumption of the peroxide. This reaction is further associated with (a) O2 evolution; (b) hydroxylation of the aromatic compound salicylate to yield a set of dihydroxybenzoic acid derivatives (analyzed by HPLC with electrochemical detection); (c) autoxidation of cholesterol with formation of 3 beta-hydroxy-5-alpha-cholest-6-ene-5-hydroperoxide; and (d) formation of electronically excited states detected by low-level chemiluminescence. The heterolytic scission of the O-O bond of hydroperoxides by metmyoglobin causes the formation of an oxidizing equivalent capable of promoting peroxidation of linoleate and arachidonate (as indicated by the parallel formation of thiobarbituric acid-reactive material and an enhancement of chemiluminescence intensity). The identity of the oxidizing equivalent(s) is discussed in terms of the formation of a relatively stable higher state of oxidation of heme Fe (FeIV-OH or FeV = O) as well as on possible intermediate species derived during the decomposition of H2O2 by metmyoglobin, such as HO.and 1O2. These species might be involved either simultaneously or sequentially in the peroxidation of fatty acids as well as in the tissue damage associated with the formation of H2O2 in ischemic-reperfusion states.     

Anthracycline antibiotic reduction by spinach ferredoxin-NADP+ reductase and ferredoxin

Spinach NADPH:ferredoxin oxidoreductase (EC 1.6.7.1) catalyzes the NADPH-dependent reduction of the anthracyclines daunomycin, aclacinomycin A, and nogalamycin and their respective 7-deoxyanthracyclinones. Under anaerobic conditions, the endogenous rate of O2 reduction by NADPH catalyzed by ferredoxin reductase (0.12 s-1 at pH 7.4) is augmented by the anthracyclines and 7-deoxyanthracyclinones. The catalytic constants are approximately equivalent for this augmentation for all substrates (approximate V of 2 s-1 and KM of 75 microM). Both O2- and H2O2 are made. Under anaerobic conditions, anthracycline reduction catalyzed by ferredoxin reductase results in the elimination of the C-7 substituent to provide a quinone methide intermediate. Following tautomerization by C-7 protonation, 7-deoxyanthracyclinones are obtained. Under appropriate conditions these may be further reduced to the 7-deoxyanthracyclinone hydroquinones. For daunomycin, the quinone methide is formed rapidly after reduction and is easily monitored at 600 nm. It may react with electrophiles other than H+, as demonstrated by its competitive trapping by p-carboxybenzaldehyde. It may also react with nucleophiles, as demonstrated by its competitive trapping by N-acetylcysteine. For aclacinomycin, quinone methide formation is also rapid although no distinct transient near 600 nm occurs. In addition to protonation, it reacts with itself providing the 7,7'-dimer. With ethyl xanthate as a thiolate nucleophile, adducts derived from addition to C-7 are obtained. For nogalamycin, quinone methide formation is not rapid. Nogalamycin is reduced to its hydroquinone, which slowly converts in a first-order process [k = (1.2 +/- 0.2) X 10(-3) s-1, pH 8.0, 30 degrees C] to the quinone methide, which is then quenched by protonation. Spinach ferredoxin in its reduced form is chemically competent for anthracycline reduction. Its effect on both the aerobic and anaerobic reactions catalyzed by ferredoxin reductase is to increase severalfold the overall velocity for anthracycline reduction. In conclusion, the aerobic reaction pathways for the anthracyclines as mediated by ferredoxin reductase are remarkably similar, while the anaerobic reactions are remarkably different. If these anthracyclines exert their antitumor activity by a common anaerobic pathway, it is most likely that the pathway is determined by the properties of the anthracycline as complexed to its in vivo target. The behavior of ferredoxin further suggests that not only low-potential flavin centers but also iron-sulfur centers should be regarded as important loci for anthracycline reductive activation.     

Reactions of glutathione and glutathione radicals with benzoquinones.

The reactions of glutathione (GSH) and glutathione radicals with a series of methyl-substituted 1,4-benzoquinones and 1,4-benzoquinone have been studied. It was found that by mixing excess benzoquinone with glutathione at pH above 6.5, the products formed were complex and unstable. All of the other experiments were carried out at pH 6.0, where the main product was stable for several hours. Stopped-flow analysis allowed the measurement of the rates of the rapid reactions between GSH and the quinones, and the products were monitored by High Performance Liquid Chromatography (HPLC). The rates of the reactions vary by five orders of magnitude and must be influenced by steric factors as well as changes in the redox states. It was observed that simple hydroquinones were not formed when the different benzoquinones were mixed with excess GSH and suggests that the initial reaction is addition/reduction rather than electron transfer. In the presence of excess quinone, the hydroquinone of the glutathione conjugate is oxidized back to its quinone. The rates of the reaction were measured. By using the technique of pulse radiolysis, it was possible to measure the reduction of the quinones by GSSG.- and the oxidation of hydroquinones by GS(.). It is proposed that the appearance of GSSG in reactions of quinones with glutathione could be due to oxidation of the hydroquinone by oxygen and the subsequent superoxide or H2O2 promoting the oxidation of GSH to GSSG.     

The involvement of iron in lipid peroxidation. Importance of ferric to ferrous ratios in initiation

Intense lipid peroxidation of brain synaptosomes initiated with Fenton's reagent (H2O2 + Fe2+) began instantly upon addition of Fe2+ and preceded detectable OH. formation. Although mannitol or Tris partially blocked peroxidation, concentrations required were 10(3)-fold in excess of OH. actually formed, and inhibition by Tris was pH dependent. Lipid peroxidation also was initiated by either Fe2+ or Fe3+ alone, although significant lag phases (minutes) and slowed reaction rates were observed. Lag phases were dramatically reduced or nearly eliminated, and reaction rates were increased by a combination of Fe3+ and Fe2+. In this instance, lipid peroxidation initiated by optimal concentrations of H2O2 and Fe2+ could be mimicked or even surpassed by providing optimal ratios of Fe3+ to Fe2+. Peroxidation observed with Fe3+ alone was dependent upon trace amounts of contaminating Fe2+ in Fe3+ preparations. Optimal ratios of Fe3+:Fe2+ for the rapid initiation of lipid peroxidation were on order of 1:1 to 7:1. No OH. formation could be detected with this system. Although low concentrations of H2O2 or ascorbate increased lipid peroxidation by Fe2+ or Fe3+, respectively, high concentrations of H2O2 or ascorbate (in excess of iron) inhibited lipid peroxidation due to oxidative or reductive maintenance of iron exclusively in Fe2+ or Fe3+ form. Stimulation of lipid peroxidation by low concentrations of H2O2 or ascorbate was due to the oxidative or reductive creation of Fe3+:Fe2+ ratios. The data suggest that the absolute ratio of Fe3+ to Fe2+ was the primary determining factor for the initiation of lipid peroxidation reactions.     

Autocatalytic quinone methide formation from mitomycin c

Mitomycin c in the presence of NADPH and brewers' yeast NADPH: (acceptor) oxidoreductase (Old Yellow enzyme, EC 1.6.99.1) is transformed, at pH 8.0 and with anaerobicity, to two major mitosene products (the cis- and trans-1-hydroxy-2,7-diaminomitosenes; respective yields of 45 and 30%). These arise by covalent trapping by solvent of a quinone methide intermediate, obtained by rearrangement of the mitomycin c hydroquinone. At lower pH (6.5), the major product of this reaction is 2,7-diaminomitosene, which arises by covalent trapping of the quinone methide by H+. In the former instance the quinone methide acts as an electrophile and in the latter as a nucleophile. A detailed kinetic analysis indicates that the role of the NADPH and Old Yellow enzyme is to initiate an autocatalytic reaction, propagated by mitomycin c reduction by the mitosene hydroquinones (arising by the electrophilic pathway). The evidence supporting this conclusion is the formation of oxidized mitosene products, under the rigorously anaerobic reaction circumstance, the nonstoichiometric participation of NADPH, a dependence of the velocity on the total mitomycin c concentration, the pH dependence of the reaction, and the accurate simulation of the overall kinetic course with a mathematical model of the autocatalytic pathway. These observations indicate that the autocatalytic pathway may be dominant during in vitro mitomycin c anaerobic reductive activation by other reducing agents and that (as with anthracycline reductive activation) oxidation of the mitosene hydroquinone obtained from nucleophile addition to the quinone methide may be a necessary event for the formation of stable covalent adducts in vivo.     

Monooxygenase activities of dioxygenases. Benzphetamine demethylation and aniline hydroxylation reactions catalyzed by indoleamine 2,3-dioxygenase

Benzphetamine demethylase and aniline hydroxylase activities were determined with various hemoproteins including indoleamine 2,3-dioxygenase in a cytochrome P-450-like reconstituted system containing NADPH, NADPH-cytochrome P-450 reductase, and O2. The highest specific activities, almost comparable to those of liver microsomal cytochrome P-450, were detected with indoleamine 2,3-dioxygenase from the rabbit intestine. The indoleamine 2,3-dioxygenase-catalyzed benzphetamine demethylation reaction was inhibited by catalase but not by superoxide dismutase. Exogenous H2O2 or organic hydroperoxides was able to replace the reducing system and O2. The stoichiometry of H2O2 added to the product formed was essentially unity. These results indicate that the dioxygenase catalyzes the demethylation reaction by the so-called "peroxygenation" mechanism using H2O2 generated in the reconstituted system. On the other hand, the dioxygenase-catalyzed aniline hydroxylation reaction was not only completely inhibited by catalase but also suppressed by superoxide dismutase by about 60%. Although the O2- and H2O2-generating system (e.g. hypoxanthine-xanthine oxidase) was also active as the reducing system, neither exogenous H2O2 nor the generation of O2- in the presence of catalase supported the hydroxylation reaction, indicating that both H2O2 and O2- were essential for the hydroxylation reaction. However, typical scavengers for hydroxyl radical and singlet oxygen were not inhibitory. These results suggest that a unique, as yet unidentified active oxygen species generated by H2O2 and O2- participates in the dioxygenase-mediated aniline hydroxylation reaction.     

Glutathionyl- and hydroxyl radical formation coupled to the redox transitions of 1,4-naphthoquinone bioreductive alkylating agents during glutathione two-electron reductive addition.

The kinetic parameters of the redox transitions subsequent to the two-electron transfer implied in the glutathione (GSH) reductive addition to 2- and 6-hydroxymethyl-1,4-naphthoquinone bioalkylating agents were examined in terms of autoxidation, GSH consumption in the arylation reaction, oxidation of the thiol to glutathione disulfide (GSSG), and free radical formation detected by the spin-trapping electron spin resonance method. The position of the hydroxymethyl substituent in either the benzenoid or the quinonoid ring differentially influenced the initial rates of hydroquinone autoxidation as well as thiol oxidation. Thus, GSSG- and hydrogen peroxide formation during the GSH reductive addition to 6-hydroxymethyl-1,4-naphthoquinone proceeded at rates substantially higher than those observed with the 2-hydroxymethyl derivative. The distribution and concentration of molecular end products, however, was the same for both quinones, regardless of the position of the hydroxymethyl substituent. The [O2]consumed/[GSSG]formed ratio was above unity in both cases, thus indicating the occurrence of autoxidation reactions other than those involved during GSSG formation. EPR studies using the spin probe 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) suggested that the oxidation of GSH coupled to the above redox transitions involved the formation of radicals of differing structure, such as hydroxyl and thiyl radicals. These were identified as the corresponding DMPO adducts. The detection of either DMPO adduct depended on the concentration of GSH in the reaction mixture: the hydroxyl radical adduct of DMPO prevailed at low GSH concentrations, whereas the thiyl radical adduct of DMPO prevailed at high GSH concentrations. The production of the former adduct was sensitive to catalase, whereas that of the latter was sensitive to superoxide dismutase as well as to catalase. The relevance of free radical formation coupled to thiol oxidation is discussed in terms of the thermodynamic and kinetic properties of the reactions involved as well as in terms of potential implications in quinone cytotoxicity.     

Photoisomerization and photo-hydration of 3-hydroxystyrylnaphthalenes.

Fluorescence and trans-->cis photoisomerization are the main deactivation paths following excitation of trans-1-(2'-naphthyl)-2-(3'-hydroxyphenyl)ethene (trans-2,3NOH) in cyclohexane, methanol and acetonitrile. The quantum yield of both processes is wavelength dependent: this is due to the presence of conformational isomers deriving from rotation of the naphthyl group around the single bond with ethene. Addition of water to acetonitrile quenches the fluorescence (lambda(max)= 380 nm). In CH(3)CN/H(2)O (4/6, v/v) the emission spectrum displays a broad band with maximum at approximately 550 nm besides the original quenched fluorescence. This indicates that 2,3-NOH undergoes acid-base equilibration in the singlet excited state as supported by the enhancement of the fluorescence quantum yield with increasing acidity of the medium. Ground and excited state acidity constants have been determined. The main photochemical process is photo-hydration, i.e. water addition to the ethene bond. Fluorescence and photo-hydration have the same sigmoidal dependence on the acid concentration, which indicates that the undissociated form of singlet excited 2,3NOH is the photoreactive species. Laser flash photolysis experiments allowed identification of the reactive intermediates. The photophysics of trans-1-(1'-naphthyl)-2-(3'-hydroxyphenyl)ethene (trans-1,3NOH) is similar to that of 2,3NOH as regards the effect of water on fluorescence and the acid-base behaviour in the ground and first excited singlet state; the main photochemical process is trans-->cis photoisomerization together with photo-cylization to hydroxychrysene in neutral water/acetonitrile, but with lower yield compared to cyclohexane, and photo-hydration in strongly acidic medium.