Free radical formation and DNA strand breakage during metabolism of diaziquone by NAD(P)H quinone-acceptor oxidoreductase (DT-diaphorase) and NADPH cytochrome c reductase.

One-electron reduction of diaziquone (AZQ) by purified rat liver NADPH cytochrome c reductase was associated with formation of AZQ semiquinone, superoxide anions, hydrogen peroxide, and hydroxyl radicals as indicated by ESR spin-trapping studies. Reactive oxygen formation correlated with AZQ-dependent production of single and double PM2 plasmid DNA strand breaks mediated by this system as detected by gel electrophoresis. Direct two-electron reduction of AZQ by purified rat liver NAD(P)H (quinone acceptor) oxidoreductase (QAO) was also associated with formation of AZQ semiquinone, superoxide anions, hydrogen peroxide, and hydroxyl radicals as detected by ESR spin trapping. Furthermore, PM2 plasmid DNA strand breaks were detected in the presence of this system. Plasmid DNA strand breakage was inhibited by dicumarol (49 +/- 5%), catalase (57 +/- 2.3%), SOD (42.2 +/- 3.6%) and ethanol (41.1 +/- 3.9%) showing QAO and reactive oxygen formation was involved in the PM2 plasmid DNA strand breaks observed. These results show that both one- and two-electron enzymatic reduction of AZQ give rise to formation of reactive oxygen species and DNA strand breaks. Autoxidation of the AZQ semiquinone and hydroquinone in the presence of molecular oxygen appears to be responsible for these processes. QAO appears to be involved in the metabolic activation of AZQ to free radical species. The cellular levels and distribution of this enzyme may play an important role in the response of tumor and normal cells to this antitumor agent.     

DNA-protein cross-linking via guanine oxidation: dependence upon protein and photosensitizer

DNA-protein cross-links form when guanine undergoes a 1-electron oxidation in a flash-quench experiment, and the importance of reactive oxygen species, protein, and photosensitizer is examined here. In these experiments, a strong oxidant produced by oxidative quenching of a DNA-bound photosensitizer generates an oxidized guanine base that reacts with protein to form the covalent adduct. These cross-links are cleaved by hot piperidine and are not the result of reactive oxygen species, since neither a hydroxyl radical scavenger (mannitol) nor oxygen affects the yield of DNA-histone cross-linking, as determined via a chloroform extraction assay. The cross-linking yield depends on protein, decreasing as histone > cytochrome c > bovine serum albumin. The yield does not depend on the cytochrome oxidation state, suggesting that reduction of the guanine radical by ferrocytochrome c does not compete effectively with cross-linking. The photosensitizer strongly influences the cross-linking yield, which decreases in the order Ru(phen)(2)dppz(2+) [phen = 1,10-phenanthroline; dppz = dipyridophenazine] > Ru(bpy)(3)(2+) [bpy = 2,2'-bipyridine] > acridine orange > ethidium, in accordance with measured oxidation potentials. A long-lived transient absorption signal for ethidium dication in poly(dG-dC) confirms that guanine oxidation is inefficient for this photosensitizer. From a polyacrylamide sequencing gel of a (32)P-labeled 40-mer, all of these photosensitizers are shown to damage guanines preferentially at the 5' G of 5'-GG-3' steps, consistent with a 1-electron oxidation. Additional examination of ethidium shows that it can generate cross-links between histone and plasmid DNA (pUC19) and that the yield depends on the quencher. Altogether, these results illustrate the versatility of the flash-quench technique as a way to generate physiologically relevant DNA-protein adducts via the oxidation of guanine and expand the scope of such cross-linking reactions to include proteins that may associate only transiently with DNA.     

Decreased skp2 expression is necessary but not sufficient for therapy-induced senescence in prostate cancer

Therapy-induced senescence (TIS), a cytostatic stress response in cancer cells, is induced inefficiently by current anticancer agents and radiation. The mechanisms that mediate TIS in cancer cells are not well defined. Herein, we characterize a robust senescence response both in vitro and in vivo to the quinone diaziquone (AZQ), previously identified in a high-throughput senescence-induction small-molecule screen. Using AZQ and several other agents that induce senescence, we screened a series of cyclin-dependent kinase inhibitors and found that p27(Kip1) was induced in all investigated prostate cancer cell lines. The ubiquitin-ligase Skp2 negatively regulates p27(Kip1) and, during TIS, is translocated to the cytoplasm before its expression is decreased in senescent cells. Overexpression of Skp2 blocks the effects of AZQ on senescence and p27(Kip1) induction. We also find that stable long-term short hairpin RNA knockdown of Skp2 decreases proliferation but does not generate the complete senescence phenotype. We conclude that Skp2 participates in regulating TIS but, alone, is insufficient to induce senescence in cancer cells.     

Characterization of insoluble elastin from copper-deficient pigs. Its usefulness in elastin sequence studies.

Insoluble elastin from copper-deficient animals has an amino acid composition intermediate between mature elastin and salt-soluble elastin (a higher lysine content and correspondingly low number of cross-links relative to the normal protein) and is solubilized by successive treatment with trypsin and chymotrypsin at 4 and 37 degrees C. Small amounts of B3H4 (11 mg--2 g of elastin) reduced allysine, allysine aldol, dehydronorleucine, and dehydromerodesmosine in insoluble elastin from copper-deficient pig aorta. In contrast, desmosine and isodesmosine were reduced only when a large excess of reductant (400 mg borohydride) was included in the reaction mixture. Reduction studies indicated that lysinonorleucine and merodesmosine were present in their dehydro forms to a greater extent in copper-deficient pig elastin than in normal elastin. After reduction with borohydride approximately 35% of the reduced form of the insoluble elastin remained insoluble after digestion with trypsin and chymotrypsin. A peptide containing the aldehyde oxidation product of lysine (allysine) and demonstrating an enrichment in glutamic acid was purified from the reduced form of copper-deficient pig elastin and partially sequenced. Its sequence (Gly-Ala-Glu-allysine-(Glu)...) and amino acid composition suggest: (1) clustering of glutamic acid residues in the elastin molecule, and (2) that allysine residues are not restricted to the alanine-enriched sites described for other elastin cross-links. Insoluble elastin from copper-deficient animals promises to be a useful tool for elastin sequence studies.     

Electron spin resonance of electrochemically generated free radicals from diaziquone and its derivatives

The one-electron electrochemical reduction of diaziquone (AZQ) and 12 analogs is analyzed using ESR spectroscopy and cyclic voltammetry. The hyperfine coupling constants arising from the interaction of the unpaired electron with the aziridine nitrogen nuclei fall within 1.20 and 2.26 G. Smaller couplings are observed arising from the protons and nitrogens in the carboethoxyamino groups. The in vitro activity of AZQ and its analogs is examined. Methyl groups in the aziridine rings increase the activity of some analogs. In the absence of aziridines, a chloroquinone compound with only carboethoxyamino groups was surprisingly active. This compound has a more positive cathodic peak than diaziquone.     

Trace detection of hydroxyl radicals during the redox cycling of low concentrations of diaziquone: a new approach

Quantifying oxygen radicals that arise during the redox cycling of quinone-containing anticancer agents such as diaziquone (AZQ) has been difficult, as has been their detection at low drug concentrations. This is due to the fact that EPR spin trapping, the method most often used for *OH detection, requires the use of high drug concentrations. Using a new highly sensitive technique that employs a fluorescamine-derivatized nitroxide, we show that low levels of NADPH-cytochrome P450 reductase (4.25 microg/ml) catalyze the production of hydroxyl radicals at very low, clinically relevant AZQ concentrations. Thus, at this enzyme concentration, we were able to detect a rate of 0.10 nM s(-1) hydroxyl radical production by 5 microM AZQ, a clinically relevant concentration. The Michaelis-Menten constants for AZQ-mediated hydroxyl radical production are: K(M) = 10.7 +/- 1.4 microM, and V(max) = 5.2 +/- 0.9 x 10(-8) M s(-1) (mg protein)(-1). Experiments employing catalase, superoxide dismutase, and NADPH-cytochrome P450 reductase, confirm the previously deduced conclusions from high drug concentrations, that is, that at low concentrations, AZQ acts to shuttle reducing equivalents from the enzyme to oxygen, thus generating the redox cycle. The data presented here suggest that the levels and locations of redox active metal ions may be the principal controlling factor in the pathway of AZQ activity that involves oxidative stress.     

Diaziquone as a potential agent for photoirradiation therapy: formation of the semiquinone and hydroxyl radicals by visible light

When diaziquone was irradiated with 500 nm visible light, hydroxyl free radicals as well as the diaziquone semiquinone were produced. The diaziquone semiquinone is a stable free radical that exhibits a characteristic 5-line electron spin resonance (ESR) spectrum. Since hydroxyl free radicals are short lived, and not observable by conventional ESR, the nitrone spin trap 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) was used to convert hydroxyl radicals into longer lived ESR detectable spin adducts. The formation of hydroxyl radicals was further confirmed by investigating reactions in which hydroxyl radical scavangers, sodium formate and dimethylsulfoxide, compete with the spin traps DMPO or POBN (alpha-(4-Pyridyl-1-oxide)-N- tert-butylnitrone) for hydroxyl free radicals. The products of these scavenging reactions were also trapped with DMPO or POBN. If drug free radicals and hydroxyl free radicals are important in the activity of quinone-containing antitumor agents, AZQ may have a potential in photoirradiation therapy or photodynamic therapy.     

Activation And Deactivation Of Quinones Catalyzed By Dt-Diaphorase. Evidence For Bioreductive Activation Of Diaziquone (Azq) In Human Tumor Cells And Detoxification Of Benzene Metabolites In Bone Marrow Stroma

Bioactivation of diaziquone (AZQ) in HT-29 human colon carcinoma cells and detoxification of benzene metabolites in bone marrow stromal cells were used as examples of the potential role of DT-diaphorase in both activation and deactivation processes. HT-29 cell cytosol contained high levels of DT-diaphorase activity and removed AZQ in the presence of either NADH or NADPH. Prior boiling of cytosol. omission of NADH or NADPH or inclusion of dicoumarol. an inhibitor of DT-diaphorase. inhibited removal of AZQ. AZQ-inouced cytotoxicity in HT-29 cells was also inhibited by dicoumarol. Chemical reduction of AZQ in a cell free system enhanced formation of a GSH conjugate of AZQ. Two of the major cell types in bone marrow stroma are macrophages and fibroblastoid stromal cells. A fibroblastoid cell line derived from stromal cells contained approximately fourfold higher levels of DT-diaphorase than macrophages. Inclusion of dicournarol in incubations containing ¹⁴C-hydroquinone and the respective stromal cell type, significantly increased covalent binding of radiolabel to macromolecules in stromal fibroblasts but not in macrophages     

Detection of oxidized lipid-modified erythrocyte membrane proteins by radiolabeling with tritiated borohydride

Human erythrocyte ghosts treated with tert-butyl hydroperoxide or ADP-Fe3+ incorporated radioactivity on reduction with tritiated borohydride. The tritium incorporation closely correlated with membrane lipid oxidation as assessed by the formation of thiobarbituric acid-reactive substances and fluorescent substances. Treatment of ghosts with the inducers in the presence of butylated hydroxytoluene, thiourea, or desferrioxamine suppressed the tritium incorporation in the subsequent reduction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the tritiated ghost proteins showed that the label was incorporated into the intermolecularly cross-linked and the uncross-linked proteins of bands 1, 2, 3, 4.1, 4.2, 5 and 6, and into the noncross-linked glycophorin A (PAS-1). Glycophorin A was hardly cross-linkable but modified during membrane lipid oxidation. Possible candidates for producing borohydride-reducible functions in the proteins are various mono- and bifunctional aldehydes, as well as those for producing fluorescence and cross-links. A part of thiobarbituric acid-reactive or fluorescent substances may be involved in borohydride reduction and tritium labeling.     

Diaziquone-induced micronuclei in cytochalasin B-blocked mouse peripheral blood lymphocytes

A mouse peripheral blood lymphocyte (PBL) micronucleus (MN) test was developed using a modification of the technique for assessing MN in human PBLs described by Fenech and Morley (1985). Male C57Bl/6 mice (5/dose) were injected i.p. with either 0, 2.5, 5.0, 7.5, or 10.0 mg diaziquone (AZQ)/kg. After 24 h the mice were bled by cardiac puncture, PBLs were isolated on a Ficoll-density gradient and then cultured in RPMI 1640 medium using 8 micrograms phytohemagglutinin/ml. In some cultures cytochalasin B (CYB) was added at 21 h during the medium change to block cytokinesis. In other cultures, CYB was omitted to compare the sensitivity of analyzing MN in binucleate versus unblocked mononucleate cells. All doses of AZQ yielded significant increases in MN-containing binucleated PBLs. The use of CYB in the mouse PBL MN test increased the sensitivity approximately 3-fold. The MN test in mouse PBLs should be useful in comparative cytogenetic studies of mice and humans.     

Structural, energetic and dynamic properties of guanine(C8)-thymine(N3) cross-links in DNA provide insights on susceptibility to nucleotide excision repair

The one-electron oxidation of guanine in DNA by carbonate radical anions, a decomposition product of peroxynitrosocarbonate which is associated with the inflammatory response, can lead to the formation of intrastrand cross-links between guanine and thymine bases [Crean et al. (Oxidation of single-stranded oligonucleotides by carbonate radical anions: generating intrastrand cross-links between guanine and thymine bases separated by cytosines. Nucleic Acids Res. 2008; 36: 742-755.)]. These involve covalent bonds between the C8 positions of guanine (G*) and N3 of thymine (T*) in 5'-d(…G*pT*…) and 5'-d(…G*pCpT*…) sequence contexts. We have performed nucleotide excision repair (NER) experiments in human HeLa cell extracts which show that the G*CT* intrastrand cross-link is excised with approximately four times greater efficiency than the G*T* cross-link embedded in 135-mer DNA duplexes. In addition, thermal melting studies reveal that both lesions significantly destabilize duplex DNA, and that the destabilization induced by the G*CT* cross-link is considerably greater. Consistent with this difference in NER, our computations show that both lesions dynamically distort and destabilize duplex DNA. They disturb Watson-Crick base-pairing and base-stacking interactions, and cause untwisting and minor groove opening. These structural perturbations are much more pronounced in the G*CT* than in the G*T* cross-link. Our combined experimental and computational studies provide structural and thermodynamic understanding of the features of the damaged duplexes that produce the most robust NER response.     

Preparation,structure, and properties of periodate-oxidised ATP,a potential affinity-labelling reagent

(1) Periodate oxidation of ATP yields a single product which has been purified and characterised. Periodate-oxidised ATP (o-ATP) behaves as a single compound during TLC analysis, but NMR spectral studies show that it exists in aqueous solution as an equilibrium mixture of three dialdehyde monohydrates and a dihydrate. Little free aldehyde is present. The dialdehyde monohydrates are in the form of diastereomeric cyclic hemiacetals. (2) The dialdehyde grouping of o-ATP can be reduced with sodium borohydride, producing a dialcohol. (3) o-ATP has been frequently used in attempts to affinity label nucleotide-binding sites on proteins. The proposed structure of o-ATP is discussed in relation to this use for o-ATP.     

Cross-linking methionine and amine residues with reactive halogen species

Irreversible cross-links are increasingly being recognized as important posttranslational oxidative protein modifications that contribute to tissue injury during oxidative stress and inflammation. They also have a structural function in extracellular matrix proteins such as collagen IV. Likely contenders for forming such cross-links are the reactive halogen species that are generated by neutrophils and eosinophils, including hypochlorous acid, hypobromous acid, and their related haloamines. Methionine residues are kinetically preferred targets for these oxidants and oxidation can potentially result in sulfilimine (>SN–) bonds with amines. Therefore, we investigated whether oxidation of methionine in the model peptide formyl-Met-Leu-Phe-Lys (fMLFK) produces cross-links with lysine residues, using mass spectrometry to characterize the products. As expected, the sulfoxide was the major product with each reactive halogen species. However, intra- and intermolecular cross-linked products were also formed. Isomers of an intramolecular sulfilimine were readily produced by hypobromous acid and bromamines, with hypochlorous acid forming lesser amounts. The predominant cross-link with chloramines was an intermolecular bond between the sulfur of fMLFK and the amine derived from the chloramine. Reactive halogen species also formed these sulfilimine cross-links in other peptides that contain methionine. We propose that protein cross-links involving methionine and amine residues will form via this mechanism when granulocytes are activated at sites of inflammation. Our results also support the proposal that reactive halogen species generated by the peroxidase peroxidasin could be responsible for the sulfilimine bonds that are integral to the structure of collagen IV.     

Chemistry of the collagen cross-links. Isolation and characterization of two intermediate intermolecular cross-links in collagen

This paper describes the isolation from reduced collagen of two new amino acids believed to be involved, in their non-reduced form, as intermolecular cross-links stabilizing the collagen fibre. The reduction of intact collagen fibrils with tritiated sodium borohydride was found to stabilize the aldehyde-mediated cross-links to acid hydrolysis and thus allowed their location and isolation from acid hydrolysates on an automatic amino acid analyser. Comparison of the radioactive elution patterns from the autoanalyser of collagen treated in various ways before reduction permitted a preliminary classification of the peaks into cross-link precursors, intramolecular and intermolecular cross-links. The techniques employed to isolate the purified components on a large scale and to identify them structurally are described in detail. Two labile intermolecular cross-links were isolated in their reduced forms, one of which was identified by high-resolution mass spectrometry as N-(5-amino-5-carboxypentyl)hydroxylysine. The structure of this compound was confirmed by chemical synthesis. The cross-link precursor α-aminoadipic δ-semialdehyde was isolated in its reduced form, -hydroxynorleucine, together with its acid degradation product -chloronorleucine. A relatively stable intermolecular cross-link was isolated and partially characterized by mass spectrometry as an aldol resulting from the reaction of the δ-semialdehyde derived from lysine and hydroxylysine.     

Mechanism(s) for the metabolism of mitoxantrone: electron spin resonance and electrochemical studies

Mitoxantrone has been reported to lack certain properties that characterize quinone containing antitumor agents that undergo enzymatic reduction. These properties are the stimulation of NADPH oxidation, the stimulation of oxygen consumption by microsomes and reductases and, the absence of oxygen free radicals during these reactions. Having these properties implies the presence of a futile redox cycle that requires the generation and the oxidation of a semiquinone free radical. It would follow that if mitoxantrone does not redox cycle in the presence of reductases, then the semiquinone free radical is not produced or, if it is formed, it reacts quickly to form diamagnetic products. However, using liver microsomes, there are reports of the formation of the mitoxantrone free radial anion. In this paper we investigated the mitoxantrone free radical anion generated electrochemically and found that in the presence of oxygen it behaved like other semiquinones. That is, it is oxidized to the parent compound (presumably generating oxygen free radicals), indicating the ability to redox cycle. The reduction potential to generate such free radical in aqueous medium is very high (-0.79 V) when compared to diaziquone (-0.36 V) and Adriamycin (-0.6 V). This suggests that mitoxantrone may not be a substrate for reductases. Under reductive conditions with purified NADPH cytochrome P-450 reductase which very easily reduces diaziquone and Adriamycin, mitoxantrone was not reduced. However, under the same conditions, mitoxantrone was oxidized by the prototype oxidase horseradish peroxidase with the production of a mitoxantrone free radical. This oxidation was accompanied by a drastic change in color and the formation of a dark precipitate. Because microsomes contain a variety of enzymes, we suggest that the previously observed free radical in microsomes is probably due to the oxidation of mitoxantrone. In this theory, this product is probably a polymer which would not require oxygen to be formed. Thus, under oxidative conditions, the mitoxantrone free radical cation will also display impaired redox activity.     

Modification of glycophorin A during oxidation of erythrocyte membrane

Human erythrocyte ghosts were oxidized with tert-butyl hydroperoxide and subsequently treated with tritiated borohydride to label the membrane proteins modified during the membrane oxidation. From the ghosts, oxidized-and-tritiated glycophorin A was isolated and characterized. No intermolecular cross-links were observed as analyzed by sodium dodecylsulfate gel electrophoresis. But, the number of lysine residues was significantly reduced and susceptibility to proteinases such as trypsin, chymotrypsin and pronase was lower than that of control glycophorin A. Trypsinization of the oxidized-and-tritiated glycophorin A gave insoluble and soluble trypsin fragments. After dansylation, N-terminal amino acids of the trypsin-fragments were determined. Dansyl amino acids from the insoluble trypsin fragments were not identical with those from control insoluble counterparts in the membrane-spanning region of glycophorin A molecule. Fractionation by gel filtration of dansyl-soluble trypsin fragments, and the N-terminal amino acid analysis of the fractionated peptides indicated that the peptides derived from the glycosylated region located in the outside of the membrane matrix were identical with those from control soluble counterparts. The results suggest that the glycosylated outside region of glycophorin A was modified only slightly but the hydrophobic membrane-spanning region was extensively modified during membrane oxidation, most likely by oxidized lipids.     

Formation of methyl ester of 2-methylglyceric acid from thymine glycol residues: a convenient new method for determining radiation damage to DNA

Thymine glycol residues in DNA or thymidine were converted to methyl 2-methylglycerate by reaction with alkaline borohydride followed by methanolic HCl. The product was labeled either from [3H]DNA or from [3H]borohydride and was followed by cochromatography with authentic 14C-labeled material. Following acid hydrolysis, the identity of 2-methylglyceric acid was confirmed by high-resolution mass spectrometry, NMR, IR, and elemental analysis. Treatment of DNA or thymidine with X-irradiation, with H2O2 and Fe2+, with H2O2, Cu2+, and ascorbate, and with H2O2 and ultraviolet light, permanganate, or sonication all produced methyl 2-methylglycerate in varying amounts after alkaline borohydride and methanolic HCl, whereas untreated DNA did not. The data indicate that certain oxidants including hydroxyl radicals generated by chemical means or from radiolysis of water convert thymine residues to thymine glycols in DNA, which can be determined as methyl 2-methylglycerate.     

Oxidatively induced DNA-protein cross-linking between single-stranded binding protein and oligodeoxynucleotides containing 8-oxo-7,8-dihydro-2'-deoxyguanosine

The formation of covalent cross-links between amino acid side chains and DNA bases in DNA-protein complexes is a significant pathway in oxidative damage to the genome, yet much remains to be learned about their chemical structures and mechanisms of formation. In the present study, DNA-protein cross-links (DPCs) were formed between synthetic oligodeoxynucleotides containing an 8-oxo-7,8-dihydro-2'deoxyguanosine (OG) or an 8-oxo-7,8-dihydro-2'-deoxyadenosine (OA) nucleotide and Escherichia coli singled-stranded binding protein (SSB) under oxidative conditions. Studies with various sequences indicated that DNA homopolymers and those lacking 8-oxopurines were less reactive toward DPC formation. DPCs were formed in the presence of HOCl, peroxynitrite, and the one-electron oxidants Na(2)IrCl(6), Na(2)IrBr(6), and Na(3)Fe(CN)(6). Protein-protein cross-linking was also observed, particularly for oxidants of high reduction potential such as Na(2)IrCl(6). The adducted oligodeoxynucleotides were sensitive to hot piperidine treatment leading to strand scission at the site of cross-linking. In addition, the covalent cross-links were somewhat heat and acid labile, which may be related to the difficulties encountered in obtaining complete characterization of trypsin digests of the DPCs. However, model reactions involving the single amino acids lysine, arginine, and tyrosine, residues known to be involved in base contacts in the DNA:SSB complex, could be studied, and the adduct formed between N(alpha)-acetyllysine methyl ester and an 18-mer containing OG was tentatively characterized by electrospray ionization mass spectrometry as analogues of spiroiminodihydantoin and guanidinohydantoin. A mechanism involving nucleophilic attack of an amino acid side chain (e.g. the epsilon-amino group of lysine) at C5 of a 2-electron oxidized form of OG is proposed.     

Mechanisms of acetaminophen oxidation to N-acetyl-P-benzoquinone imine by horseradish peroxidase and cytochrome P-450

Horseradish peroxidase rapidly catalyzed the H2O2-dependent polymerization of acetaminophen. Acetaminophen polymerization was decreased and formation of GSSG and minor amounts of GSH-acetaminophen conjugates were detected in reaction mixtures containing GSH. These data suggest that horseradish peroxidase catalyzed the 1-electron oxidation of acetaminophen and that GSH decreased polymerization by reducing the product, N-acetyl-p-benzosemiquinone imine, back to acetaminophen. Analyses of reaction mixtures that did not contain GSH showed N-acetyl-p-benzoquinone imine formation shortly after initiation of reactions. When GSH was added to similar reaction mixtures at various times, 3-(glutathion-S-yl)-acetaminophen was formed. The formation and disappearance of this product were very similar to N-acetyl-p-benzoquinone imine formation and were consistent with the disproportionation of 2 mol of N-acetyl-p-benzosemiquinone imine to 1 mol of N-acetyl-p-benzoquinone imine and 1 mol of acetaminophen followed by the rapid reaction of N-acetyl-p-benzoquinone imine with GSH to form 3-(glutathion-S-yl)acetaminophen. When acetaminophen was incubated with NADPH, oxygen and hepatic microsomes from phenobarbital-pretreated rats, 1.2 nmol 3-(glutathion-S-yl)acetaminophen/nmol cytochrome P-450/10 min was formed. Formation of polymers was not observed indicating that N-acetyl-p-benzoquinone imine was formed via an overall 2-electron oxidation rather than a disproportionation reaction. However, when cumene hydroperoxide was replaced by NADPH in microsomal incubations, polymerization was observed suggesting that cytochrome P-450 might also catalyze the 1-electron oxidation of acetaminophen.     

Phenoxazinone synthase: mechanism for the formation of the phenoxazinone chromophore of actinomycin

Phenoxazinone synthase is a copper-containing oxidase that catalyzes the coupling of 2-aminophenols to form the 2-aminophenoxazinone chromophore. This reaction constitutes the final step in the biosynthesis of the potent antineoplastic agent actinomycin. The mechanism of this complex 6-electron oxidation was determined by using a variety of substituted 2-aminophenols, designed to block the reaction at intermediate stages. Thus, with 3,5-di-tert-butyl-2-aminophenol as substrate, the reaction was blocked at the o-quinone imine 17; with 5-tert-butyl-2-aminophenol (19) as substrate, the reaction was blocked at the p-quinone imine 20; and with 5-methyl-2-aminophenol (21) as substrate, the reaction was blocked at the dihydro-2-aminophenoxazinone 22. These findings suggested a mechanism in which 2-aminophenoxazinone formation proceeded via a quinone imine intermediate 4 that was trapped by a second molecule of 2-aminophenol. Oxidation of the adduct 5 to the p-quinone imine 6 was followed by a second conjugate addition and a final 2-electron oxidation to give the product, 2-aminophenoxazinone. The role of the enzyme in the catalysis of each of these steps was examined. It was found that the second conjugate addition generated a racemic center at C4a, suggesting that this reaction did not occur at the active site. A deuterium isotope effect on the cleavage of the C4-H bond of 2-aminophenol suggested that partial dissociation of an intermediate from the enzyme occurred after the first conjugate addition. It is proposed that 2-aminophenoxazinone synthesis proceeds via a sequence of three consecutive 2-electron aminophenol oxidations and that the aminophenol moiety is regenerated during the reaction sequence by facile tautomerization reactions. Thus, what initially appears to be an impressively complex mechanism may, in fact, be ingeniously simple.