Two-electron Reduction Of Quinones Research Articles

Iron deficiency aggravates DMNQ-induced cytotoxicity via redox cycling in kidney-derived cells

Iron, an essential element for most of living organisms, participates in many biological functions. Since iron is redox-active transition metal, it is known that excessive levels stimulate the formation of reactive oxygen species (ROS) and exacerbate cytotoxicity. An iron deficiency is the most common nutritional deficiency disorder in the world (about 30% of the population) and is more common than cases of iron overload. However, the effects of iron deficiency on ROS-induced cytotoxicity and the maintenance of intracellular redox homeostasis are not fully understood. The present study reports on an evaluation of the effects of iron deficiency on cytotoxicity induced by several ROS generators. In contrast to hydrogen peroxide and erastin, the cytotoxicity of 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), a redox cycling agent that induces intracellular superoxide anion formation, was exacerbated by iron deficiency. Cytochrome b5 reductase was identified as a candidate enzyme responsible for the redox cycling of DMNQ under conditions of iron depletion. Moreover, the DMNQ-induced intracellular accumulation of ROS and a decrease in NADH/NAD+ ratios were enhanced by an iron deficiency. These negative changes were found to be ameliorated by overexpressing NAD(P)H:quinone oxidoreductase 1 (NQO1) in kidney-derived cells that originally showed a very low expression of NQO1. These results indicate that NQO1 plays a protective role against redox cycling quinone-mediated cytotoxicity under iron-depleted conditions. This is because NQO1 generates less-toxic hydroquinones via the two-electron reduction of quinones. The collective findings reported herein demonstrate that not only an iron overload but also an iron deficiency exacerbates ROS-mediated cytotoxicity.

Quinone- and nitroreductase reactions of Thermotoga maritima thioredoxin reductase.

The Thermotoga maritima NADH:thioredoxin reductase (TmTR) contains FAD and a catalytic disulfide in the active center, and uses a relatively poorly studied physiological oxidant Grx-1-type glutaredoxin. In order to further assess the redox properties of TmTR, we used series of quinoidal and nitroaromatic oxidants with a wide range of single-electron reduction potentials (E(1)7, -0.49-0.09 V). We found that TmTR catalyzed the mixed single- and two-electron reduction of quinones and nitroaromatic compounds, which was much faster than the reduction of Grx-1. The reactivity of both groups of oxidants increased with an increase in their E(1)7, thus pointing to the absence of their structural specificity. The maximal rates of quinone reduction in the steady-state reactions were lower than the maximal rates of reduction of FAD by NADH, obtained in presteady-state experiments. The mixed-type reaction inhibition by NAD(+) was consistent with its competition for a NADH binding site in the oxidized enzyme form, and also with the reoxidation of the reduced enzyme form. The inhibition data yielded a value of the standard potential for TmTR of -0.31±0.03 V at pH 7.0, which may correspond to the FAD/FADH2 redox couple. Overall, the mechanism of quinone- and nitroreductase reactions of T. maritima TR was similar to the previously described mechanism of Arabidopsis thaliana TR, and points to their prooxidant and possibly cytotoxic role.

Abstract 1388: Biological role of novel NQO2 inhibitors in cancer therapeutics

Abstract Background: NRH: quinone oxidoreductase 2 (NQO2), is a cytosolic flavoprotein that catalyses the two-electron reduction of quinones into hydroquinones. The physiological role of NQO2 in cancer biology is still not well understood. Some studies have suggested that inhibiting NQO2 could have a great potential in chemoprevention. This is based on the observation that NQO2 gene knockdown promotes protection against the toxicity of quinones such as menadione. Aim: To evaluate the biological role of novel NQO2 inhibitors in cancer therapeutics. Novel inhibitors will be used as pharmacological tools to study the physiological role of NQO2 in cancer cells, and to identify the consequences of inhibiting NQO2 on the tumor microenvironment. Methods: Novel compounds with diverse structures have been tested for their inhibition of NQO2 enzymatic activity in a cell-free system using a pure enzyme assay. The functional activity of these compounds as NQO2 inhibitors in the MDA-MB 231 breast cancer cells was also assessed. The effect of these inhibitors on the cellular proliferation of MDA-MB 231 breast cancer cells was tested using an MTT assay. Off-target (DNA binding) effects were analyzed by measuring the DNA thermal temperature. Moreover, the effect of these inhibitors on modulating TNFα-mediated NF-kB transcriptional activity was tested and the mechanism behind this modulation was also examined. Results: The novel inhibitors tested resulted in the inhibition of NQO2 activity in the cell free and the cellular system at nano-molar concentrations .Inhibition of MDA-MB 231 cell proliferation was also witnessed at low micro-molar concentrations. The inhibitors also displayed no DNA binding ability. In addition, the inhibitors were able to attenuate TNFα-mediated NF-kB transcriptional activity by blocking the phosphorylation of IkBα. This has resulted in the suppression of cell growth and promotion of cell apoptosis. Conclusions: This study has demonstrated novel potent inhibitors of NQO2 that are functionally active in the cells at nano-molar concentrations. The effect of the inhibitors on TNFα-mediated NF-kB transcriptional activity raises the possibility that inhibiting NQO2 could modulate the tumor micro-environmental factors, such as metastasis and angiogenesis. Citation Format: Elham Santina, Amy Chadwick, Soraya Al Nabulsi, Sally Freeman, Constantinos Demonacos, Ian Stratford. Biological role of novel NQO2 inhibitors in cancer therapeutics. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1388. doi:10.1158/1538-7445.AM2014-1388

Evidence for NQO1 and NQO2 catalyzed reduction of ortho- and para-quinone methides

NAD(P)H:quinone oxidoreductase (NQO1) and NRH:quinone oxidoreductase 2 (NQO2) catalyze the two-electron reduction of quinones and thereby prevent generation of toxic radicals. Quinone methides (QMs) covalently react with cellular macromolecules to form DNA adducts and/or protein conjugates resulting in toxicity and carcinogenesis. Based on similar structural features of quinones and QMs, it is logical to assume that NQO1 and/or NQO2 could also catalyze the two-electron reduction of QMs. However, hitherto the reduction of QMs, as both endogenous and/or exogenous biological substrates, by either NQO1/NQO2 has never been demonstrated. Here we show for the first time that both NQO1 and NQO2 can catalyze the reduction of electrophilic ortho-/para-QMs. The involvement of the enzyme in the reduction of p-cresol quinone methide (PCQM) and o-cresol quinone methide (OCQM) was demonstrated by reappearance of NQO1/NQO2-FAD peak at 450 nm after addition of the QMs to the assay mixture. Further reduction of methides by NQO1/NQO2 was confirmed by analyzing the assay mixture by tandem mass spectrometry. Preliminary kinetic studies show that NQO2 is faster in reducing QMs than its homolog NQO1, and moreover, ortho-QMs are reduced faster than para-QMs. Enzyme-substrate docking studies showed results consistent with enzyme catalysis. Thus, NQO1/NQO2 can play a significant role in deactivation of QMs.

Abstract 3061: Colocalization of NQO1 with the mitotic spindle in human cells

Abstract NAD(P)H:quinone oxidoreductase 1 (NQO1) is a flavoenzyme that catalyzes the two-electron reduction of quinones. NQO1 has also been shown to reduce superoxide to hydrogen peroxide and regulate ubiquitin independent 20S proteasome degradation of proteins such as p53. In normal tissues NQO1 is expressed primarily in epithelium, endothelium and adipocytes where it is believed to play a protective role. NQO1 is also expressed at relatively high levels in many human solid tumors where it has been targeted for its ability to bioactivate anti-tumor quinones. Interestingly, immunohistochemical staining for NQO1 expression in formalin-fixed human non-small cell lung cancer tissue sections revealed strong staining for NQO1 on the mitotic spindles of cells in mitosis. Immunocytochemical studies in combination with confocal microscopy using human nontransformed and transformed cells (HUVEC, HBMEC, astrocytes) as well as human cancer cell lines confirmed NQO1 staining of mitotic spindles. Staining of mitotic spindles for NQO1 was not observed in cells genotyped as homozygous for the NQO1*2 polymorphism or in cells pretreated with siRNA directed against NQO1. Double-labeling experiments using anti-alpha-tubulin and anti-NQO1 antibodies demonstrated colocalization of NQO1 with spindle microtubules. Pretreatment with NQO1 inhibitors (ES936 or dicumarol) did not prevent binding of NQO1 to mitotic spindles suggesting that binding is independent of the redox state of the enzyme. In cells not undergoing mitosis there was no apparent association of NQO1 with the cytoskeleton suggesting that binding of NQO1 to microtubules may be specific for mitotic spindles. Previous immunocytochemical studies by other investigators have shown that p53 and 20S proteasome could also be found in association with the mitotic spindle. Whether NQO1 binds directly to spindle microtubules or the binding of NQO1 to the spindle is dependent upon the association with other proteins such as p53 or 20S proteasome is currently under investigation (Supported by NIH grant R01ES018943). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3061. doi:1538-7445.AM2012-3061

ATP independent proteasomal degradation of NQO1 in BL cell lines

Human NAD(P)H: quinone oxidoreductase 1 (NQO1) catalyzes the obligatory two-electron reduction of quinones. For this peculiar catalytic mechanism, the enzyme is considered an important cytoprotector. The NQO1 gene is expressed in all human tissues, unless a polymorphism due to C609T point mutation is present. This polymorphism produces a null phenotype in the homozygous condition and reduced enzyme activity in the heterozygous one. We previously demonstrated that two cell lines of haematopoietic origin, HL60 and Raji cells, possess the same heterozygous genotype, but different phenotypes; as expected for a heterozygous condition the HL60 cell line showed a low level of enzyme activity, while the Raji cell line appeared as null phenotype. The level of NQO1 mRNA was similar in the two cell lines and the different phenotype was not due to additional mutations or to expression of alternative splicing products. Here we show that in Raji BL cell line with heterozygous genotype the null NQO1 phenotype is due to 20S proteasome degradation of wild type and mutant protein isoforms and is not directly linked to C609T polymorphism. This finding may have important implications in B-cell differentiation, in leukaemia risk evaluation and in chemotherapy based on proteasome inhibitors.

Abstract 5471: Mechanism-based inhibition of NRH: quinone oxidoreductase 2 (NQO2) by a series of aminoalkylamino substituted indolequinones

Abstract NQO2 is an NRH-dependent quinone reductase that catalyzes the two-electron reduction of quinones to their corresponding hydroquinones. Despite structural similarities between NAD(P)H quinone oxidoreductase 1 (NQO1) and NQO2 many compounds have been shown to bind selectively to NQO2. Previous studies have shown that many drugs including resveratrol, quercetin, chloroquine, and imatinib can bind to and inhibit the catalytic activity of NQO2. Whether the binding and inhibition of NQO2 influences the pharmacology of these compounds is currently unknown. In this study we have developed a series of indolequinones as mechanism-based inhibitors of NQO2. These compounds were based on ES936 (5-methoxy-1,2-dimethyl-3-(4-nitrophenoxy)methylindole-4,7-dione) an efficient mechanism-based inhibitor of NQO1. We found that substitution of the 5-methoxy group in ES936 with an aminoalkylamino side chain generated potent mechanism-based inhibitors of NQO2 that do not efficiently inhibit NQO1. Interestingly, MD436 (5-amino-1,2-dimethyl-3-(2,4,6-trifluorophenoxy)methylindole-4,7-dione) a compound that contains only an amino group at the 5-position was a mechanism-based inhibitor of NQO1 and NQO2. Structural requirements for optimal inhibition of NQO2 were examined and found to include an aminoalkylamino side chain at the 5-position. NQO2 inhibition was observed with compounds with alkyl chains that varied from 3 to 8 carbons in length, with maximal inhibition observed with compounds that possessed chain lengths of between 3 to 5 carbons. Compounds where the aminoalkyl chain terminated in either an unsubstituted amino group or a dimethyl substituted amino group were efficient inhibitors of NQO2. Compounds with substitutions at the 6-position were not efficient mechanism-based inhibitors of NQO2 despite molecular docking studies that suggested NQO2 could accommodate either 5 or 6-position aminoalkylamino side chains. Co-crystallization studies are currently underway to more precisely define the physical interactions between compounds substituted at the 5-position and NQO2. As previously observed for a series of NQO1 mechanism-based inhibitors, structural requirements for efficient inhibition of NQO2 also include a methyl group at the 2-position of the indole ring and a substituent at the 3-position capable of leaving to generate a reactive iminium electrophile following enzymatic reduction. MD372 (5-(3-aminopropylamino)-1,2-dimethyl-3-(2,4,6-trifluorophenoxy)methylindole-4,7-dione) was tested for NQO2 inhibition using the human leukemia cell line K562 that has been shown to contain very high levels of NQO2 protein and activity. Treatment of K562 cells with MD372 (100nM) for 15 min resulted in greater than 95% inhibition of NQO2 catalytic activity, confirming that these compounds enter cells and efficiently inhibit NQO2 (Supported by R21 HL095995). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5471.

WrbA from Escherichia coli and Archaeoglobus fulgidus Is an NAD(P)H:Quinone Oxidoreductase

WrbA (tryptophan [W] repressor-binding protein) was discovered in Escherichia coli, where it was proposed to play a role in regulation of the tryptophan operon; however, this has been put in question, leaving the function unknown. Here we report a phylogenetic analysis of 30 sequences which indicated that WrbA is the prototype of a distinct family of flavoproteins which exists in a diversity of cell types across all three domains of life and includes documented NAD(P)H:quinone oxidoreductases (NQOs) from the Fungi and Viridiplantae kingdoms. Biochemical characterization of the prototypic WrbA protein from E. coli and WrbA from Archaeoglobus fulgidus, a hyperthermophilic species from the Archaea domain, shows that these enzymes have NQO activity, suggesting that this activity is a defining characteristic of the WrbA family that we designate a new type of NQO (type IV). For E. coli WrbA, the K(m)(NADH) was 14 +/- 0.43 microM and the K(m)(benzoquinone) was 5.8 +/- 0.12 microM. For A. fulgidus WrbA, the K(m)(NADH) was 19 +/- 1.7 microM and the K(m)(benzoquinone) was 37 +/- 3.6 microM. Both enzymes were found to be homodimeric by gel filtration chromatography and homotetrameric by dynamic light scattering and to contain one flavin mononucleotide molecule per monomer. The NQO activity of each enzyme is retained over a broad pH range, and apparent initial velocities indicate that maximal activities are comparable to the optimum growth temperature for the respective organisms. The results are discussed and implicate WrbA in the two-electron reduction of quinones, protecting against oxidative stress.

FAD semiquinone stability regulates single- and two-electron reduction of quinones by Anabaena PCC7119 ferredoxin:NADP + reductase and its Glu301Ala mutant

Flavoenzymes may reduce quinones in a single-electron, mixed single- and two-electron, and two-electron way. The mechanisms of two-electron reduction of quinones are insufficiently understood. To get an insight into the role of flavin semiquinone stability in the regulation of single- vs. two-electron reduction of quinones, we studied the reactions of wild type Anabaena ferredoxin:NADP +reductase (FNR) with 48% FAD semiquinone (FADH ) stabilized at the equilibrium (pH 7.0), and its Glu301Ala mutant (8% FADH at the equilibrium). We found that Glu301Ala substitution does not change the quinone substrate specificity of FNR. However, it confers the mixed single- and two-electron mechanism of quinone reduction (50% single-electron flux), whereas the wild type FNR reduces quinones in a single-electron way. During the oxidation of fully reduced wild type FNR by tetramethyl-1,4-benzoquinone, the first electron transfer (formation of FADH ) is about 40 times faster than the second one (oxidation of FADH ). In contrast, the first and second electron transfer proceeded at similar rates in Glu301Ala FNR. Thus, the change in the quinone reduction mechanism may be explained by the relative increase in the rate of second electron transfer. This enabled us to propose the unified scheme of single-, two- and mixed single- and two-electron reduction of quinones by flavoenzymes with the central role of the stability of flavin/quinone ion–radical pair.

Structure-Activity Relationships in Two-Electron Reduction of Quinones

Publisher Summary This chapter analyzes the structure-activity relationships in two-electron reduction of quinines. Quinones may accept electrons from various flavoenzymes, iron-sulfur proteins and photosynthetic reaction centers. The energetics of the quinine reduction are studied extensively by pulse-radiolysis, electron spin resonance, and electrochemical techniques. The single-electron reduction of quinones by flavoenzyme dehydrogenases-electrontransferases may be treated according to an ‘‘outer-sphere electron transfer’’ model. In general, the reaction rates increase with an increase in quinine single-electron reduction potential. In this chapter, it is demonstrated that multiparameter regression analysis, by using redox potential and several simple structural parameters of quinones, may provide important information on the mechanisms of two-electron enzymatic reduction. In view of the simplicity of the single-electron reduction mechanism and the presumable involvement of single-electron transfers in two-electron reduction, the single-electron reduction of quinones is analyzed. Single-electron reduction of quinones by flavoenzymes is analyzed. The chapter presents the outer-sphere electron transfer model in single-electron reduction of quinones. Structure-activity relationships in single-electron reduction of quinones by NADPH:Cytochrome P-450 reductase and ferredoxin:NADP + reductase are explored. Two-electron reduction of quinones by flavoenzymes is explained in the chapter. The chapter describes the mechanism of two-electron (Hydride) transfer.

Interactions of Quinones with Thioredoxin Reductase: A CHALLENGE TO THE ANTIOXIDANT ROLE OF THE MAMMALIAN SELENOPROTEIN

Mammalian thioredoxin reductases (TrxR) are important selenium-dependent antioxidant enzymes. Quinones, a wide group of natural substances, human drugs, and environmental pollutants may act either as TrxR substrates or inhibitors. Here we systematically analyzed the interactions of TrxR with different classes of quinone compounds. We found that TrxR catalyzed mixed single- and two-electron reduction of quinones, involving both the selenium-containing motif and a second redox center, presumably FAD. Compared with other related pyridine nucleotide-disulfide oxidoreductases such as glutathione reductase or trypanothione reductase, the k(ca)(t)/K(m) value for quinone reduction by TrxR was about 1 order of magnitude higher, and it was not directly related to the one-electron reduction potential of the quinones. A number of quinones were reduced about as efficiently as the natural substrate thioredoxin. We show that TrxR mainly cycles between the four-electron reduced (EH(4)) and two-electron reduced (EH(2)) states in quinone reduction. The redox potential of the EH(2)/EH(4) couple of TrxR calculated according to the Haldane relationship with NADPH/NADP(+) was -0.294 V at pH 7.0. Antitumor aziridinylbenzoquinones and daunorubicin were poor substrates and almost inactive as reversible TrxR inhibitors. However, phenanthrene quinone was a potent inhibitor (approximate K(i) = 6.3 +/- 1 microm). As with other flavoenzymes, quinones could confer superoxide-producing NADPH oxidase activity to mammalian TrxR. A unique feature of this enzyme was, however, the fact that upon selenocysteine-targeted covalent modification, which inactivates its normal activity, reduction of some quinones was not affected, whereas that of others was severely impaired. We conclude that interactions with TrxR may play a considerable role in the complex mechanisms underlying the diverse biological effects of quinones.

Two-electron reduction of quinones by Enterobacter cloacae NAD(P)H:nitroreductase: quantitative structure-activity relationships

Enterobacter cloacae NAD(P)H:nitroreductase (NR; EC 1.6.99.7) catalyzes two-electron reduction of a series of quinoidal compounds according to a “ping-pong” scheme, with marked substrate inhibition by quinones. The steady-state catalytic constants ( k cat) range from 0.1 to 1600 s −1, and bimolecular rate constants ( k cat/ K m) range from 10 3 to 10 8 M −1 s −1 . Quinones, nitroaromatic compounds and competitive to NADH inhibitor dicumarol, quench the flavin mononucleotide (FMN) fluorescence of nitroreductase. The reactivity of NR with single-electron acceptors is consistent with an “outer-sphere” electron transfer model, taking into account high potential of FMN semiquinone/FMNH − couple and good solvent accessibility of FMN. However, the single-electron acceptor 1,1 ′-dibenzyl-4,4 ′-bipyridinium was far less reactive than quinones possessing similar single-electron reduction potentials (E 1 7). For all quinoidal compounds except 2-hydroxy-1,4-naphthoquinones, there existed parabolic correlations between the log of rate constants of quinone reduction and their E 1 7 or hydride-transfer potential (E 7(Q/QH −)). Based on pH dependence of rate constants, a single-step hydride transfer seems to be a more feasible quinone reduction mechanism. The reactivities of 2-hydroxy-1,4-naphthoquinones were much higher than expected from their reduction potential. Most probably, their enhanced reactivity was determined by their binding at or close to the binding site of NADH and dicumarol, whereas other quinones used the alternative, currently unidentified binding site.

Quercetin may act as a cytotoxic prooxidant after its metabolic activation to semiquinone and quinoidal product

In the last ten years, there has been an important increase in interest in quercetin action as a unique antioxidant, but its putative role in numerous prooxidant effects is also being continually updated. The mechanism underlying this undesirable ability seems to involve its metabolic oxidoreductive activation. Based on the structural properties of quercetin, we have investigated whether its catechol moiety may be the potential tool for revealed toxicity. We demonstrated, with an ESR spin-stabilization technique coupled to conventional spectrophotometry, that o-semiquinone and o-quinone are indeed the products of enzymatically catalyzed oxidative degradation of quercetin. The former radical might serve to facilitate the formation of superoxide and depletion of GSH, which could confer a specificity of its prooxidative action in situ. The observed one-electron reduction of o-quinone may enrich the semiquinone pool, thereby magnifying its effect. The two-electron reduction of quinone can result in constant resupply of quercetin in situ, thereby also modulating another pathway of its known biological activities. We have also tried to see whether the intracellular oxidative degradation of quercetin can be confirmed under the controlled conditions of model monolayer cell cultures. The results are indicative of the intracellular metabolic activation of quercetin to o-quinone, the process which can be partially associated with the observed concentration-dependent cytotoxic effect of quercetin.

One- and two-electron reduction of 2-methyl-1,4-naphthoquinone bioreductive alkylating agents: kinetic studies, free-radical production, thiol oxidation and DNA-strand-break formation

The one- and two-electron enzymic reduction of the bioreductive alkylating agents 2-methylmethoxynaphthoquinone (quinone I) and 2-chloromethylnaphthoquinone (quinone II) was studied with purified NADPH-cytochrome P-450 reductase and DT-diaphorase respectively, and characterized in terms of kinetic constants, oxyradical production, thiol oxidation and DNA-strand-break formation. The catalytic-centre activity values indicated that DT-diaphorase catalysed the reduction of quinone I far more efficiently than NADPH-cytochrome P-450 reductase, although the Km values of the two enzymes for this quinone were similar (1.2-3.0 microM). The one-electron-transfer flavoenzyme also catalysed the reduction of quinone II, but the behaviour of DT-diaphorase towards this quinone did not permit calculation of kinetic constants. A salient feature of the redox transitions caused by the one- and two-electron catalysis of these quinones was the different contributions of disproportionation and autoxidation reactions respectively. In the former case, about 26% of NADPH consumed was accounted for in terms of autoxidation (as H2O2 formation), whereas in the latter, the autoxidation component accounted for most (98%) of the NADPH consumed. This difference was abrogated by superoxide dismutase, which enhanced autoxidation during NADPH-cytochrome P-450 catalysis to a maximal value. E.s.r. analysis indicated the formation of superoxide radicals, the signal of which was suppressed by superoxide dismutase and unaffected by catalase. The one- and two-electron reduction of these quinones in the presence of GSH was accompanied by formation of thiyl radicals. Although superoxide dismutase suppressed the thiol radical e.s.r. signal in both instances, the enzyme enhanced GSSG accumulation during NADPH-cytochrome P-450 catalysis of quinone I, whereas it inhibited GSSG formation during reduction of the quinone by DT-diaphorase. One- and two-electron reduction of quinone I led to calf thymus DNA-strand-break formation, a process that (a) was substantially decreased in experiments performed with dialysed DNA and in the presence of desferal and (b) was partially sensitive to superoxide dismutase and/or catalase. These findings are rationalized in terms of the occurrence of metal ions ligated to DNA, protecting against the toxic effects of superoxide radicals generated during enzymic reduction of quinones.

One- and Two-Electron Reduction of Quinones by Rat Liver Subcellular Fractions

NAD(P)H-quinone (menadione, Trolox C quinone, and alpha-tocopherol quinone) reductase activity of rat liver subcellular fractions was observed optically at 340-400 nm, and oxygen radical generation was demonstrated using the ESR spin trap, 5,5'-dimethyl-1-pyrroline-1-oxide. NAD(P)H-menadione reductase activity of the fractions decreased in the order: cytosol > microsomes > plasma membranes. Although more than 65% of the activity of microsomes and plasma membranes was inhibited on the addition of dicoumarol, no change in the menadione-mediated formation of oxygen radicals by either fraction was observed. As judged from the intensity of ESR signals, the menadione-mediated oxygen radical formation by plasma membranes was only one-tenth as great as that by microsomes. No generation of oxygen radicals in the NAD(P)H-menadione reductase reaction by cytosol was found, and the activity was abolished in the presence of dicoumarol, an inhibitor of DT-diaphorase. It is concluded that plasma membranes reduce quinones by way of two-electron transfer and that the activity may prevent cellular quinone toxicity. NAD(P)H-alpha-tocopherol quinone reductase activity was confirmed in all cellular fractions [Hayashi et al. (1992) Biochem. Pharmacol. 44, 489-493] and this activity was also inhibited by dicoumarol, suggesting that it was due to DT-diaphorase.

The mechanism of the quinone reductase reaction of pig heart lipoamide dehydrogenase

The relationship between the NADH:lipoamide reductase and NADH:quinone reductase reactions of pig heart lipoamide dehydrogenase (EC 1.6.4.3) was investigated. At pH 7.0 the catalytic constant of the quinone reductase reaction (kcat.) is 70 s-1 and the rate constant of the active-centre reduction by NADH (kcat./Km) is 9.2 x 10(5) M-1.s-1. These constants are almost an order lower than those for the lipoamide reductase reaction. The maximal quinone reductase activity is observed at pH 6.0-5.5. The use of [4(S)-2H]NADH as substrate decreases kcat./Km for the lipoamide reductase reaction and both kcat. and kcat./Km for the quinone reductase reaction. The kcat./Km values for quinones in this case are decreased 1.85-3.0-fold. NAD+ is a more effective inhibitor in the quinone reductase reaction than in the lipoamide reductase reaction. The pattern of inhibition reflects the shift of the reaction equilibrium. Various forms of the four-electron-reduced enzyme are believed to reduce quinones. Simple and 'hybrid ping-pong' mechanisms of this reaction are discussed. The logarithms of kcat./Km for quinones are hyperbolically dependent on their single-electron reduction potentials (E1(7]. A three-step mechanism for a mixed one-electron and two-electron reduction of quinones by lipoamide dehydrogenase is proposed.

One- and two-electron reduction of quinones by glutathione reductase

Yeast glutathione reductase (E.C. 1.6.4.2) catalyzes the oxidation of NADPH by p-quinones and ferricyanide with a maximal turnover number (TNmax) of 4-5 s-1.NADP+ stimulates the reaction and the TNmax/Km value of acceptors is reached at NADP+/NADPH greater than or equal to 100. TNmax is increased up to 30-33 s-1. The stimulatory effect of NADP+ may be associated with its complexation with the NADPH-binding site in the reduced enzyme (Kd = 40-60 microM). It is suggested that NADP+ shifts the electron density towards FAD in the two-electron-reduced enzyme and, evidently, changes its one-electron-reduction potentials, while quinones oxidize an equilibrium form of glutathione reductase containing reduced FAD. In the absence of NADP+ the reduction of quinones by glutathione reductase proceeds mainly in a two-electron manner. At NADP+/NADPH = 100 a one-electron reduction makes up 44% of the total process. At pH 6.0-7.0 the reduced forms of naphthoquinones undergo cyclic redox conversions. A hyperbolic dependence exists of the log TN/Km of quinones on their one-electron-reduction potentials.