Novel quinolinequinone antitumor agents: structure-metabolism studies with NAD(P)H:quinone oxidoreductase (NQO1).

The effects of functional group changes on the metabolism of novel quinolinequinones by recombinant human NAD(P)H:quinone oxidoreductase (NQO1) are described. Overall, the quinolinequinones were much better substrates for NQO1 than analogous indolequinones, with compounds containing heterocyclic substituents at C-2 being among the best substrates.     

Benzofuran-, benzothiophene-, indazole- and benzisoxazole-quinones: Excellent substrates for NAD(P)H:quinone oxidoreductase 1

A series of heterocyclic quinones based on benzofuran, benzothiophene, indazole and benzisoxazole has been synthesized, and evaluated for their ability to function as substrates for recombinant human NAD(P)H:quinone oxidoreductase (NQO1), a two-electron reductase upregulated in tumor cells. Overall, the quinones are excellent substrates for NQO1, approaching the reduction rates observed for menadione.     

Role of NAD(P)H:quinone oxidoreductase (NQO1) in apoptosis induction by aziridinylbenzoquinones RH1 and MeDZQ

We aimed to characterize the role of NAD(P)H:quinone oxidoreductase (NQO1) in apoptosis induction by antitumour quinones RH1 (2,5-diaziridinyl-3-hydroxymethyl-6-methyl-1,4-benzoquinone) and MeDZQ (2,5-dimethyl-3,6-diaziridinyl-1,4-benzoquinone). Digitonin-permeabilized FLK cells catalyzed NADPH-dependent single- and two-electron reduction of RH1 and MeDZQ. At equitoxic concentrations, RH1 and MeDZQ induced apoptosis more efficiently than the nonalkylating duroquinone or H(2)O(2). The antioxidant N,N'-diphenyl-p-phenylene diamine, desferrioxamine, and the inhibitor of NQO1 dicumarol, protected against apoptosis induction by all compounds investigated, but to a different extent. The results of multiparameter regression analysis indicate that RH1 and MeDZQ most likely induce apoptosis via NQO1-linked formation of alkylating species but not via NQO1-linked redox cycling.     

Cytotoxicity of RH1 and related aziridinylbenzoquinones: involvement of activation by NAD(P)H:quinone oxidoreductase (NQO1) and oxidative stress

It is supposed that the main cytotoxicity mechanism of antitumour aziridinyl-substituted benzoquinones is their two-electron reduction to alkylating products by NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2). However, other possible cytotoxicity mechanisms, e.g., oxidative stress, are studied insufficiently. In the single-electron reduction of quinones including a novel compound RH1 (2,5-diaziridinyl- 3-(hydroxymethyl)-6-methyl-1,4-benzoquinone), by NADPH:cytochrome P-450 reductase (EC 1.6.2.4, P-450R), their reactivity increased with an increase in the redox potential of quinone/semiquinone couple (E(1)7), reaching a limiting value at E(1)7> or =-0.1V. The reactivity of quinones towards NQO1 did not depend on their E(1)7. The cytotoxicity of aziridinyl-unsubstituted quinones in bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) mimics their reactivity in P-450R-catalyzed reactions, exhibiting a parabolic dependence on their E(1)7. The toxicity of aziridinyl-benzoquinones, although being higher, also followed this trend and did not depend on their reactivity towards NQO1. The action of aziridinylbenzoquinones in FLK cells was accompanied by an increase in lipid peroxidation, their toxicity decreased by desferrioxamine and the antioxidant N,N'-diphenyl-p-phenylene diamine, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea. The inhibitor of NQO1, dicumarol, protected against the toxicity of aziridinyl-benzoquinones except of 2,5-bis-(2'-hydroxyethylamino)-3,6-diaziridinyl-1,4-benzoquinone (BZQ), which was almost inactive as NQO1 substrate. The same events except the absence of pronounced effect of dicumarol were characteristic in the cytotoxicity of aziridinyl-unsubstituted quinones. These findings indicate that in addition to the activation by NQO1, the oxidative stress presumably initiated by single-electron transferring enzymes may be an important factor in the cytotoxicity of aziridinylbenzoquinones. The information obtained may contribute to the understanding of the molecular mechanisms of aziridinylquinone cytotoxicity and may be useful in the design of future bioreductive drugs.     

NRH:quinone oxidoreductase2 (NQO2)

The quinone oxidoreductases [NAD(P)H:quinone oxidoreductase1 (NQO1) and NRH:quinone oxidoreductase2 (NQO2)] are flavoproteins. NQO1 is known to catalyse metabolic detoxification of quinones and protect cells from redox cycling, oxidative stress and neoplasia. NQO2 is a 231 amino acid protein (25956 mw) that is 43 amino acids shorter than NQO1 at its carboxy-terminus. The human NQO2 cDNA and protein are 54 and 49% similar to the human liver cytosolic NQO1 cDNA and protein. Recent studies have revealed that NQO2 differs from NQO1 in its cofactor requirement. NQO2 uses dihydronicotinamide riboside (NRH) rather than NAD(P)H as an electron donor. Another difference between NQO1 and NQO2 is that NQO2 is resistant to typical inhibitors of NQO1, such as dicoumarol, Cibacron blue and phenindone. Flavones, including quercetin and benzo(a)pyrene, are known inhibitors of NQO2. Even though overlapping substrate specificities have been observed for NQO1 and NQO2, significant differences exist in relative affinities for the various substrates. Analysis of the crystal structure of NQO2 revealed that NQO2 contains a specific metal binding site, which is not present in NQO1. The human NQO2 gene has been precisely localized to chromosome 6p25. The human NQO2 gene locus is highly polymorphic. The NQO2 gene is ubiquitously expressed and induced in response to TCDD. Nucleotide sequence analysis of the NQO2 gene promoter revealed the presence of several cis-elements, including SP1 binding sites, CCAAT box, xenobiotic response element (XRE) and an antioxidant response element (ARE). The complement of these elements regulates tissue specific expression and induction of the NQO2 gene in response to xenobiotics and antioxidants. The in vivo role of NQO2 and its role in quinone detoxification remains unknown.     

Characterization and partial purification of microsomal NAD(P)H:quinone oxidoreductases

Quinone oxidoreductases are flavoproteins that catalyze two-electron reduction and detoxification of quinones. This leads to the protection of cells against toxicity, mutagenicity, and cancer due to exposure to environmental and synthetic quinones and its precursors. Two cytosolic forms of quinone oxidoreductases [NAD(P)H:quinone oxidoreductase 1 (NQO1) and NRH:quinone oxidoreductase 2 (NQO2)] were previously identified, purified, and cloned. A role of cytosolic NQO1 in protection of cells from oxidative stress, cytotoxicity, and mutagenicity of quinones was established. Currently, we have characterized and partially purified the NQO activity from rat liver microsomes. This activity was designated as microsomal NQO (mNQO). The mNQO activity showed significantly higher affinity for NADH than NADPH as electron donors and catalyzed reduction of 2,6-dichlorophenolindophenol and menadione. The mNQO activity was insensitive to dicoumarol, a potent inhibitor of cytosolic NQO1. Western analysis of microsomal proteins revealed 29- and 18-kDa bands that cross-reacted with polyclonal antibodies raised against cytosolic NQO1. The mNQO activity was partially purified by solubilization of microsomes with detergent Chaps, ammonium sulfate fractionation, and DEAE-Sephacel column chromatography. The microsomal mNQO proteins are expected to provide additional protection after cytosolic NQOs against quinone toxicity and mutagenicity.     

Iron (III) reduction: A novel activity of the human NAD(P)H:oxidoreductase

NAD(P)H:quinone oxidoreductase (NQO1; EC 1.6.99.2) catalyzes a two-electron transfer involved in the protection of cells from reactive oxygen species. These reactive oxygen species are often generated by the one-electron reduction of quinones or quinone analogs. We report here on the previously unreported Fe(III) reduction activity of human NQO1. Under steady state conditions with Fe(III) citrate, the apparent Michaelis-Menten constant (Km(app)) was approximately 0.3 nM and the apparent maximum velocity (Vmax(app)) was 16 U mg(-1). Substrate inhibition was observed above 5 nM. NADH was the electron donor, Km(app)= 340 microM and Vmax(app) = 46 Umg(-1). FAD was also a cofactor with a Km(app) of 3.1 microM and Vmax(app) of 89 U mg(-1). The turnover number for NADH oxidation was 25 s(-1). Possible physiological roles of the Fe(III) reduction by this enzyme are discussed.     

Implications of NQO1 in cancer therapy

NAD(P)H:quinone oxidoreductase (NQO1), an obligatory two-electron reductase, is a ubiquitous cytosolic enzyme that catalyzes the reduction of quinone substrates. The NQO1- mediated two-electron reduction of quinones can be either chemoprotection/detoxification or a chemotherapeutic response, depending on the target quinones. When toxic quinones are reduced by NQO1, they are conjugated with glutathione or glucuronic acid and excreted from the cells. Based on this protective effect of NQO1, the use of dietary compounds to induce the expression of NQO1 has emerged as a promising strategy for cancer prevention. On the other hand, NQO1-mediated two-electron reduction converts certain quinone compounds (such as mitomycin C, E09, RH1 and β-lapachone) to cytotoxic agents, leading to cell death. It has been known that NQO1 is expressed at high levels in numerous human cancers, including breast, colon, cervix, lung, and pancreas, as compared with normal tissues. This implies that tumors can be preferentially damaged relative to normal tissue by cytotoxic quinone drugs. Importantly, NQO1 has been shown to stabilize many proteins, including p53 and p33ING1b, by inhibiting their proteasomal degradation. This review will summarize the biological roles of NQO1 in cancer, with emphasis on recent findings and the potential of NQO1 as a therapeutic target for the cancer therapy. [BMB Reports 2015; 48(11): 609-617]     

NAD(P)H:Quinone Oxidoreductase 1 (NQO1) Localizes to the Mitotic Spindle in Human Cells

NAD(P)H:quinone oxidoreductase 1 (NQO1) is an FAD containing quinone reductase that catalyzes the 2-electron reduction of a broad range of quinones. The 2-electron reduction of quinones to hydroquinones by NQO1 is believed to be a detoxification process since this reaction bypasses the formation of the highly reactive semiquinone. NQO1 is expressed at high levels in normal epithelium, endothelium and adipocytes as well as in many human solid tumors. In addition to its function as a quinone reductase NQO1 has been shown to reduce superoxide and regulate the 20 S proteasomal degradation of proteins including p53. Biochemical studies have indicated that NQO1 is primarily located in the cytosol, however, lower levels of NQO1 have also been found in the nucleus. In these studies we demonstrate using immunocytochemistry and confocal imaging that NQO1 was found associated with mitotic spindles in cells undergoing division. The association of NQO1 with the mitotic spindles was observed in many different human cell lines including nontransformed cells (astrocytes, HUVEC) immortalized cell lines (HBMEC, 16HBE) and cancer (pancreatic adenocarcinoma, BXPC3). Confocal analysis of double-labeling experiments demonstrated co-localization of NQO1with alpha-tubulin in mitotic spindles. In studies with BxPc-3 human pancreatic cancer cells the association of NQO1 with mitotic spindles appeared to be unchanged in the presence of NQO1 inhibitors ES936 or dicoumarol suggesting that NQO1 can associate with the mitotic spindle and still retain catalytic activity. Analysis of archival human squamous lung carcinoma tissue immunostained for NQO1 demonstrated positive staining for NQO1 in the spindles of mitotic cells. The purpose of this study is to demonstrate for the first time the association of the quinone reductase NQO1 with the mitotic spindle in human cells.     

Genetic evidence for NAD(P)H:quinone oxidoreductase 1-catalyzed quinone reduction on passage through the mouse pulmonary circulation

The quinones duroquinone (DQ) and coenzyme Q(1) (CoQ(1)) and quinone reductase inhibitors have been used to identify reductases involved in quinone reduction on passage through the pulmonary circulation. In perfused rat lung, NAD(P)H:quinone oxidoreductase 1 (NQO1) was identified as the predominant DQ reductase and NQO1 and mitochondrial complex I as the CoQ(1) reductases. Since inhibitors have nonspecific effects, the goal was to use Nqo1-null (NQO1(-)/(-)) mice to evaluate DQ as an NQO1 probe in the lung. Lung homogenate cytosol NQO1 activities were 97 ± 11, 54 ± 6, and 5 ± 1 (SE) nmol dichlorophenolindophenol reduced·min(-1)·mg protein(-1) for NQO1(+/+), NQO1(+/-), and NQO1(-/-) lungs, respectively. Intact lung quinone reduction was evaluated by infusion of DQ (50 μM) or CoQ(1) (60 μM) into the pulmonary arterial inflow of the isolated perfused lung and measurement of pulmonary venous effluent hydroquinone (DQH(2) or CoQ(1)H(2)). DQH(2) efflux rates for NQO1(+/+), NQO1(+/-), and NQO1(-/-) lungs were 0.65 ± 0.08, 0.45 ± 0.04, and 0.13 ± 0.05 (SE) μmol·min(-1)·g dry lung(-1), respectively. DQ reduction in NQO1(+/+) lungs was inhibited by 90 ± 4% with dicumarol; there was no inhibition in NQO1(-/-) lungs. There was no significant difference in CoQ(1)H(2) efflux rates for NQO1(+/+) and NQO1(-/-) lungs. Differences in DQ reduction were not due to differences in lung dry weights, wet-to-dry weight ratios, perfusion pressures, perfused surface areas, or total DQ recoveries. The data provide genetic evidence implicating DQ as a specific NQO1 probe in the perfused rodent lung.     

NQO1-dependent redox cycling of idebenone: effects on cellular redox potential and energy levels

Short-chain quinones are described as potent antioxidants and in the case of idebenone have already been under clinical investigation for the treatment of neuromuscular disorders. Due to their analogy to coenzyme Q10 (CoQ10), a long-chain quinone, they are widely regarded as a substitute for CoQ10. However, apart from their antioxidant function, this provides no clear rationale for their use in disorders with normal CoQ10 levels. Using recombinant NAD(P)H:quinone oxidoreductase (NQO) enzymes, we observed that contrary to CoQ10 short-chain quinones such as idebenone are good substrates for both NQO1 and NQO2. Furthermore, the reduction of short-chain quinones by NQOs enabled an antimycin A-sensitive transfer of electrons from cytosolic NAD(P)H to the mitochondrial respiratory chain in both human hepatoma cells (HepG2) and freshly isolated mouse hepatocytes. Consistent with the substrate selectivity of NQOs, both idebenone and CoQ1, but not CoQ10, partially restored cellular ATP levels under conditions of impaired complex I function. The observed cytosolic-mitochondrial shuttling of idebenone and CoQ1 was also associated with reduced lactate production by cybrid cells from mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) patients. Thus, the observed activities separate the effectiveness of short-chain quinones from the related long-chain CoQ10 and provide the rationale for the use of short-chain quinones such as idebenone for the treatment of mitochondrial disorders.     

Nucleotide and deduced amino acid sequence of a human cDNA (NQO2) corresponding to a second member of the NAD(P)H:quinone oxidoreductase gene family. Extensive polymorphism at the NQO2 gene locus on chromosome 6.

NAD(P)H:quinone oxidoreductases (NQOs) are flavoproteins that catalyze the oxidation of NADH or NADPH by various quinones and oxidation-reduction dyes. We have previously described a complementary DNA that encodes a dioxin-inducible cytosolic form of human NAD(P)H:quinone oxidoreductase (NQO1). In the present report we describe the nucleotide sequence and deduced amino acid sequence for a cDNA clone that is likely to encode a second form of NAD(P)H:quinone oxidoreductase (NQO2) which was isolated by screening a human liver cDNA library by hybridization with a NQO1 cDNA probe. The NQO2 cDNA is 976 nucleotides long and encodes a protein of 231 amino acids (Mr = 25,956). The human NQO2 cDNA and protein are 54% and 49% similar to human liver cytosolic NQO1 cDNA and protein, respectively. COS1 cells transfected with NQO2 cDNA showed a 5-7-fold increase in NAD(P)H:quinone oxidoreductase activity as compared to nontransfected cells when either 2,6-dichlorophenolindophenol or menadione was used as substrate. Western blot analysis of the expressed NQO1 and NQO2 cDNA proteins showed cross-reactivity with rat NQO1 antiserum, indicating that NQO1 and NQO2 proteins are immunologically related. Northern blot analysis shows the presence of one NQO2 mRNA of 1.2 kb in control and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treated human hepatoblastoma Hep-G2 cells and that TCDD treatment does not lead to enhanced levels of NQO2 mRNA as it does for NQO1 mRNA. Southern blot analysis of human genomic DNA suggests the presence of a single gene approximately 14-17 kb in length. The NQO2 gene locus is highly polymorphic as indicated by several restriction fragment length polymorphisms detected with five different restriction enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two-electron reduction of quinones by rat liver NAD(P)H:quinone oxidoreductase: quantitative structure-activity relationships

Mammalian NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2) catalyzes the two-electron reduction of quinones and plays one of the main roles in the bioactivation of quinoidal drugs. In order to understand the enzyme substrate specificity, we have examined the reactions of rat NQO1 with a number of quinones with available potentials of single-electron (E(1)(7)) reduction and pK(a) of their semiquinones. The hydride transfer potentials (E(7)(H(-))) were calculated from the midpoint potentials of quinones and pK(a) of hydroquinones. Our findings imply that benzo- and naphthoquinones with a van der Waals volume (VdWvol) < or = 200 A(3) are much more reactive than glutathionyl-substituted naphthoquinones, polycyclic quinones, and FMN (VdWvol>200 A(3)) with the same reduction potentials. The entropies of activation (DeltaS(not equal)) in the reduction of "fast" oxidants are equal to -84 to -76 J mol(-1) K(-1), whereas in the reduction of "slow" oxidants Delta S(not equal)=-36 to -11 J mol(-1) K(-1). The large negative Delta S(not equal) in the reduction of fast oxidants may be explained by their better electronic coupling with reduced FAD or the formation of charge-transfer complexes, since fast oxidants bind at the dicumarol binding site, whereas the binding of some slow oxidants outside it has been demonstrated. The reactivity of quinones may be equally well described in terms of the three-step (e(-),H(+),e(-)) hydride transfer, using E(1)(7), pK(a)(QH*), and VdWvol as correlation parameters, or in terms of single-step (H(-)) hydride transfer, using E(7)(H(-)) and VdWvol in the correlation. The analysis of NQO1 reactions with single-electron acceptors and quinones using an "outer-sphere" electron transfer model points to the possibility of a three-step hydride transfer.     

The human dioxin-inducible NAD(P)H: quinone oxidoreductase cDNA-encoded protein expressed in COS-1 cells is identical to diaphorase 4

NAD(P)H: quinone oxidoreductase (NQO1) is believed to be protective against cancer and toxicity caused by exposure to quinones and their metabolic precursors. This enzyme catalyzes the two-electron reduction of compounds, compared with one-electron reduction mediated by NADPH: cytochrome-P450 oxidoreductase which produces toxic and mutagenic free radicals. Recently we cloned and sequenced the cDNA encoding human 2.3,7,8-tetrachlorodibenzo-p-dioxin (dioxin)-inducible cytosolic NQO1 [Jaiswal et al. (1988) J. Biol. Chem. 263, 13572-13578] and provided preliminary evidence that this enzyme may correspond to diaphorase 4, an enzymatic activity present in various tissues that catalyzes the reduction of a variety of quinones by both NADH and NADPH [Edwards et al. (1980) Biochem. J. 187, 429-436]. In the present report we characterize the catalytic properties of the protein encoded by the NQO1 cDNA. The enzyme was synthesized in monkey kidney COS-1 cells transfected with a pMT2-based expression plasmid containing the NQO1 cDNA. Western blot analysis of the transfected cells using an antibody against rat liver cytosolic NQO1 revealed a 31-kDa band that was not detected in nontransfected cells. This band corresponded to a polypeptide with the same electrophoretic mobility as the endogenous NQO1 protein detected in the human hepatoblastoma (Hep-G2) cells with the same antibody. The immunoreactive protein detected in human Hep-G2 cells was induced approximately fourfold by exposure of the cultures to dioxin, an increase commensurate with the increased in quinone oxidoreductase activity. These studies suggest that the protein encoded by NQO1 cDNA is indeed similar, if not identical, to the dioxin-inducible protein band detected in human Hep-G2 cells. Further characterization of the product of NQO1 cDNA, which was present at approximately 20-30-fold higher levels in transfected COS cells than the endogenous product in uninduced human Hep-G2 cells indicated that it had very high capacity (greater than 1000-fold over background) to catalyze the reduction of 2.6-dichloroindophenol and menadione. Besides these two commonly used substrates for quinone reductase, the expressed NQO1 protein also effectively metabolized 2,6-dimethylbenzoquinone, methylene blue, p-benzoquinone, 1,4-naphthoquinone, 2-methyl-1,4-benzoquinone, with the latter being the most potent electron acceptor at 50 microM concentration of the substrate.     

NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1), a multifunctional antioxidant enzyme and exceptionally versatile cytoprotector

NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1) is a widely-distributed FAD-dependent flavoprotein that promotes obligatory 2-electron reductions of quinones, quinoneimines, nitroaromatics, and azo dyes, at rates that are comparable with NADH or NADPH. These reductions depress quinone levels and thereby minimize opportunities for generation of reactive oxygen intermediates by redox cycling, and for depletion of intracellular thiol pools. NQO1 is a highly-inducible enzyme that is regulated by the Keap1/Nrf2/ARE pathway. Evidence for the importance of the antioxidant functions of NQO1 in combating oxidative stress is provided by demonstrations that induction of NQO1 levels or their depletion (knockout, or knockdown) are associated with decreased and increased susceptibilities to oxidative stress, respectively. Furthermore, benzene genotoxicity is markedly enhanced when NQO1 activity is compromised. Not surprisingly, human polymorphisms that suppress NQO1 activities are associated with increased predisposition to disease. Recent studies have uncovered protective roles for NQO1 that apparently are unrelated to its enzymatic activities. NQO1 binds to and thereby stabilizes the important tumor suppressor p53 against proteasomal degradation. Indeed, NQO1 appears to regulate the degradative fate of other proteins. These findings suggest that NQO1 may exercise a selective “gatekeeping” role in regulating the proteasomal degradation of specific proteins, thereby broadening the cytoprotective role of NQO1 far beyond its highly effective antioxidant functions.     

Reduction of nitroaromatic compounds by NAD(P)H:quinone oxidoreductase (NQO1): the role of electron-accepting potency and structural parameters in the substrate specificity

We aimed to elucidate the role of electronic and structural parameters of nitroaromatic compounds in their two-electron reduction by NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2). The multiparameter regression analysis shows that the reactivity of nitroaromatic compounds (n=38) increases with an increase in their single-electron reduction potential and the torsion angle between nitrogroup(s) and the aromatic ring. The binding efficiency of nitroaromatics in the active center of NQO1 exerted a less evident role in their reactivity. The reduction of nitroaromatics is characterized by more positive entropies of activation than the reduction of quinones. This points to a less efficient electronic coupling of nitroaromatics with the reduced isoalloxazine ring of FAD, and may explain their lower reactivity as compared to quinones. Another important but poorly understood factor enhancing the reactivity of nitroaromatics is their ability to bind at the dicumarol/quinone binding site in the active center of NQO1.     

Evidence for NQO2-mediated reduction of the carcinogenic estrogen ortho-quinones

The physiological function of NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase) is to detoxify potentially reactive quinones by direct transfer of two electrons. A similar detoxification role has not been established for its homologue NRH:quinone oxidoreductase 2 (NQO2). Estrogen quinones, including estradiol(E(2))-3,4-Q, generated by estrogen metabolism, are thought to be responsible for estrogen-initiated carcinogenesis. In this investigation, we have shown for the first time that NQO2 catalyzes the reduction of electrophilic estrogen quinones and thereby may act as a detoxification enzyme. ESI and MALDI mass spectrometric binding studies involving E(2)-3,4-Q with NQO2 clearly support the formation of an enzyme-substrate physical complex. The problem of spontaneous reduction of substrate by cofactor, benzyldihydronicotinamide riboside (BNAH), was successfully overcome by taking advantage of the ping-pong mechanism of NQO2 catalysis. The involvement of the enzyme in the reduction of E(2)-3,4-Q was further supported by addition of the inhibitor quercetin to the assay mixture. NQO2 is a newly discovered binding site (MT3) of melatonin. However, addition of melatonin to the assay mixture did not affect the catalytic activity of NQO2. Preliminary kinetic studies show that NQO2 is faster in reducing estrogen quinones than its homologue NQO1. Both UV and liquid chromatography-tandem mass spectrometry assays unequivocally corroborate the reduction of estrogen ortho-quinones by NQO2, indicating that it could be a novel target for prevention of breast cancer initiation.     

Two-electron reduction of quinones by Enterobacter cloacae PB2 pentaerythritol tetranitrate reductase: quantitative structure-activity relationships

In order to clarify the poorly understood mechanisms of two-electron reduction of quinones by flavoenzymes, we examined the quinone reductase reactions of a member of a structurally distinct old yellow enzyme family, Enterobacter cloacae PB2 pentaerythritol tetranitrate reductase (PETNR). PETNR catalyzes two-electron reduction of quinones according to a 'ping-pong' scheme. A multiparameter analysis shows that the reactivity of quinones increases with an increase in their single-electron reduction potential and pK(a) of their semiquinones (a three-step (e(-),H(+),e(-)) hydride transfer scheme), or with an increase in their hydride-transfer potential (E(7)(H(-))) (a single-step (H(-)) hydride transfer scheme), and decreases with a decrease in their van der Waals volume. However, the pH-dependence of PETNR reactivity is more consistent with a single-step hydride transfer. A comparison of X-ray data of PETNR, mammalian NAD(P)H : quinone oxidoreductase (NQO1), and Enterobacter cloacae nitroreductase, which reduce quinones in a two-electron way, and their reactivity revealed that PETNR is much less reactive, and much less sensitive to the quinone substrate steric effects than NQO1. This may be attributed to the lack of pi-pi stacking between quinone and the displaced aromatic amino acid in the active center, e.g., with Phe-178' in NQO1.