One-electron reduction of quinones by the neuronal nitric-oxide synthase reductase domain

Flavin electron transferases can catalyze one- or two-electron reduction of quinones including bioreductive antitumor quinones. The recombinant neuronal nitric oxide synthase (nNOS) reductase domain, which contains the FAD-FMN prosthetic group pair and calmodulin-binding site, catalyzed aerobic NADPH-oxidation in the presence of the model quinone compound menadione (MD), including antitumor mitomycin C (Mit C) and adriamycin (Adr). Calcium/calmodulin (Ca2+/CaM) stimulated the NADPH oxidation of these quinones. The MD-mediated NADPH oxidation was inhibited in the presence of NAD(P)H:quinone oxidoreductase (QR), but Mit C- and Adr-mediated NADPH oxidations were not. In anaerobic conditions, cytochrome b5 as a scavenger for the menasemiquinone radical (MD*-) was stoichiometrically reduced by the nNOS reductase domain in the presence of MD, but not of QR. These results indicate that the nNOS reductase domain can catalyze a only one-electron reduction of bivalent quinones. In the presence or absence of Ca2+/CaM, the semiquinone radical species were major intermediates observed during the oxidation of the reduced enzyme by MD, but the fully reduced flavin species did not significantly accumulate under these conditions. Air-stable semiquinone did not react rapidly with MD, but the fully reduced species of both flavins, FAD and FMN, could donate one electron to MD. The intramolecular electron transfer between the two flavins is the rate-limiting step in the catalytic cycle [H. Matsuda, T. Iyanagi, Biochim. Biophys. Acta 1473 (1999) 345-355). These data suggest that the enzyme functions between the 1e- <==> 3e- level during one-electron reduction of MD, and that the rates of quinone reductions are stimulated by a rapid electron exchange between the two flavins in the presence of Ca2+/CaM.     

Structure-based development of anticancer drugs: complexes of NAD(P)H:quinone oxidoreductase 1 with chemotherapeutic quinones

BACKGROUND: NAD(P)H:quinone acceptor oxidoreductase (QR1) protects animal cells from the deleterious and carcinogenic effects of quinones and other electrophiles. Remarkably, the same enzyme activates cancer prodrugs that become cytotoxic only after two-electron reduction. QR1's ability to bioactivate quinones and its elevated expression in many human solid tumors makes this protein an excellent target for enzyme-directed drug development. Until now, structural analysis of the mode of binding of chemotherapeutic compounds to QR1 was based on model building using the structures of complexes with simple substrates; no structure of complexes of QR1 with chemotherapeutic prodrugs had been reported. RESULTS: Here we report the high-resolution crystal structures of complexes of QR1 with three chemotherapeutic prodrugs: RH1, a water-soluble homolog of dimethylaziridinylbenzoquinone; EO9, an aziridinylindolequinone; and ARH019, another aziridinylindolequinone. The structures, determined to resolutions of 2.0 A, 2.5 A, and 1.86 A, respectively, were refined to R values below 21% with excellent geometry. CONCLUSIONS: The structures show that compounds can bind to QR1 in more than one orientation. Surprisingly, the two aziridinylindolequinones bind to the enzyme in different orientations. The results presented here reveal two new factors that must be taken into account in the design of prodrugs targeted for activation by QR1: the enzyme binding site is highly plastic and changes to accommodate binding of different substrates, and homologous drugs with different substituents may bind to QR1 in different orientations. These structural insights provide important clues for the optimization of chemotherapeutic compounds that utilize this reductive bioactivation pathway.     

Structures of mammalian cytosolic quinone reductases

The metabolism of quinone compounds presents one source of oxidative stress in mammals, as many pathways proceed by mechanisms that generate reactive oxygen species as by-products. One defense against quinone toxicity is the enzyme NAD(P)H:quinone oxidoreductase type 1 (QR1), which metabolizes quinones by a two-electron reduction mechanism, thus averting production of radicals. QR1 is expressed in the cytoplasm of many tissues, and is highly inducible. A closely related homologue, quinone reductase type 2 (QR2), has been identified in several mammalian species. QR2 is also capable of reducing quinones to hydroquinones, but unlike QR1, cannot use NAD(P)H. X-ray crystallographic studies of QR1 and QR2 illustrate that despite their different biochemical properties, these enzymes have very similar three-dimensional structures. In particular, conserved features of the active sites point to the close relationship between these two enzymes.     

Kinetic and docking studies of the interaction of quinones with the quinone reductase active site

NAD(P)H/quinone acceptor oxidoreductase type 1 (QR1) protects cells from cytotoxic and neoplastic effects of quinones though two-electron reduction. Kinetic experiments, docking, and binding affinity calculations were performed on a series of structurally varied quinone substrates. A good correlation between calculated and measured binding affinities from kinetic determinations was obtained. The experimental and theoretical studies independently support a model in which quinones (with one to three fused aromatic rings) bind in the QR1 active site utilizing a pi-stacking interaction with the isoalloxazine ring of the FAD cofactor.     

Old and new inhibitors of quinone reductase 2

Quinone reductase 2 is a cytosolic enzyme which catalyses the reduction of quinones, such as menadione and coenzymes Q. Despite a relatively close sequence-based resemblance to NAD(P)H:quinone oxidoreductase 1 (QR1), it has many different features. QR2 is the third melatonin binding site (MT3). It is inhibited in the micromolar range by melatonin, and does not accept conventional phosphorylated nicotinamides as hydride donors. QR2 has a powerful capacity to activate quinones leading to unexpected toxicity situations. In the present paper, we report the characterization of three QR2 modulators: melatonin, resveratrol and S29434. The latter compound inhibits QR2 activity with an IC₅₀ in the low nanomolar range. The potency of the modulators ranged as follows, from the least to the most potent: melatonin < resveratrol < S29434. These molecular tools might permit to explore and better understand the relationship existing between QR2 catalytic activity and the various pathological situations in which QR2 has a key role.     

NAD(P)H:(Quinone-Acceptor) Oxidoreductase of Tobacco Leaves Is a Flavin Mononucleotide-Containing Flavoenzyme

The soluble NAD(P)H:(quinone-acceptor) oxidoreductase [NAD(P)H-QR, EC 1.6.99.2] of Nicotiana tabacum L. leaves and roots has been purified. NAD(P)H-QR contains noncovalently bound flavin mononucleotide. Pairs of subunits of 21.4 kD are linked together by disulfide bridges, but the active enzyme is a homotetramer of 94 to 100 kD showing an isoelectric point of 5.1. NAD(P)H-QR is a B-stereospecific dehydrogenase. NADH and NADPH are electron donors of similar efficiency with Kcat:Km ratios (with duroquinone) of 6.2 x 107 and 8.0 x 107 m-1 s-1, respectively. Hydrophilic quinones are good electron acceptors, although ferricyanide and dichlorophenolindophenol are also reduced. The quinones are converted to hydroquinones by an obligatory two-electron transfer. No spectral evidence for a flavin semiquinone was detected following anaerobic photoreduction. Cibacron blue and 7-iodo-acridone-4-carboxylic acid are inhibitory. Tobacco NAD(P)H-QR resembles animal DT-diaphorase in some respects (identical reaction mechanism with a two-electron transfer to quinones, unusually high catalytic capability, and donor and acceptor substrate specificity), but it differs from DT-diaphorase in molecular structure, flavin cofactor, stereospecificity, and sensitivity to inhibitors. As in the case with DT-diaphorase in animals, the main NAD(P)H-QR function in plant cells may be the reduction of quinones to quinols, which prevents the production of semiquinones and oxygen radicals. The enzyme appears to belong to a widespread group of plant and fungal flavoproteins found in different cell compartments that are able to reduce quinones.     

Persuasive evidence that quinone reductase type 1 (DT diaphorase) protects cells against the toxicity of electrophiles and reactive forms of oxygen

An extensive body of evidence supports the conclusion that by catalyzing obligatory two-electron reductions of quinones to hydroquinones, NAD(P)H:quinone reductase (QR1) protects cells against the deleterious effects of redox cycling of quinones, their ability to deplete glutathione, and to produce neoplasia. The effects of elevation of QR1 levels by various enzyme inducers, inhibition of the enzyme by dicumarol, and genetic deletion of the enzyme (knockout mouse) are all consistent with the proposed protective functions. Measurement of QR1 activity in murine hepatoma cells grown in 96-well microtiter plates has provided a rapid and quantitative method for detecting inducer activity and determining inducer potency. This constitutes a strategy for the identification of potential chemoprotectors against cancer. Epidemiological studies show that humans who are genetically deficient in QR1 are more susceptible to the hematological toxicity and carcinogenicity of benzene exposure, and may be more susceptible to the development of a number of malignant tumors.     

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.     

Catalytic mechanism of 2-hydroxybiphenyl 3-monooxygenase, a flavoprotein from Pseudomonas azelaica HBP1

2-Hydroxybiphenyl 3-monooxygenase (EC 1.14.13.44) from Pseudomonas azelaica HBP1 is an FAD-dependent aromatic hydroxylase that catalyzes the conversion of 2-hydroxybiphenyl to 2, 3-dihydroxybiphenyl in the presence of NADH and oxygen. The catalytic mechanism of this three-substrate reaction was investigated at 7 degrees C by stopped-flow absorption spectroscopy. Various individual steps associated with catalysis were readily observed at pH 7.5, the optimum pH for enzyme turnover. Anaerobic reduction of the free enzyme by NADH is a biphasic process, most likely reflecting the presence of two distinct enzyme forms. Binding of 2-hydroxybiphenyl stimulated the rate of enzyme reduction by NADH by 2 orders of magnitude. The anaerobic reduction of the enzyme-substrate complex involved the formation of a transient charge-transfer complex between the reduced flavin and NAD(+). A similar transient intermediate was formed when the enzyme was complexed with the substrate analog 2-sec-butylphenol or with the non-substrate effector 2,3-dihydroxybiphenyl. Excess NAD(+) strongly stabilized the charge-transfer complexes but did not give rise to the appearance of any intermediate during the reduction of uncomplexed enzyme. Free reduced 2-hydroxybiphenyl 3-monooxygenase reacted rapidly with oxygen to form oxidized enzyme with no appearance of intermediates during this reaction. In the presence of 2-hydroxybiphenyl, two consecutive spectral intermediates were observed which were assigned to the flavin C(4a)-hydroperoxide and the flavin C(4a)-hydroxide, respectively. No oxygenated flavin intermediates were observed when the enzyme was in complex with 2, 3-dihydroxybiphenyl. Monovalent anions retarded the dehydration of the flavin C(4a)-hydroxide without stabilization of additional intermediates. The kinetic data for 2-hydroxybiphenyl 3-monooxygenase are consistent with a ternary complex mechanism in which the aromatic substrate has strict control in both the reductive and oxidative half-reaction in a way that reactions leading to substrate hydroxylation are favored over those leading to the futile formation of hydrogen peroxide. NAD(+) release from the reduced enzyme-substrate complex is the slowest step in catalysis.     

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]     

An NADH:quinone oxidoreductase active during biodegradation by the brown-rot basidiomycete Gloeophyllum trabeum

The brown-rot basidiomycete Gloeophyllum trabeum uses a quinone redox cycle to generate extracellular Fenton reagent, a key component of the biodegradative system expressed by this highly destructive wood decay fungus. The hitherto uncharacterized quinone reductase that drives this cycle is a potential target for inhibitors of wood decay. We have identified the major quinone reductase expressed by G. trabeum under conditions that elicit high levels of quinone redox cycling. The enzyme comprises two identical 22-kDa subunits, each with one molecule of flavin mononucleotide. It is specific for NADH as the reductant and uses the quinones produced by G. trabeum (2,5-dimethoxy-1,4-benzoquinone and 4,5-dimethoxy-1,2-benzoquinone) as electron acceptors. The affinity of the reductase for these quinones is so high that precise kinetic parameters were not obtainable, but it is clear that k(cat)/K(m) for the quinones is greater than 10(8) M(-1) s(-1). The reductase is encoded by a gene with substantial similarity to NAD(P)H:quinone reductase genes from other fungi. The G. trabeum quinone reductase may function in quinone detoxification, a role often proposed for these enzymes, but we hypothesize that the fungus has recruited it to drive extracellular oxyradical production.     

Differential regulation of wheat quinone reductases in response to powdery mildew infection

At least two types of quinone reductases are present in plants: (1) the ζ-crystallin-like quinone reductases (QR1, EC 1.6.5.5) that catalyze the univalent reduction of quinones to semiquinone radicals, and (2) the DT-diaphorase-like quinone reductases (QR2, EC 1.6.99.2) that catalyze the divalent reduction of quinones to hydroquinones. QR2s protect cells from oxidative stress by making the quinones available for conjugation, thereby releasing them from the superoxide-generating one electron redox cycling, catalyzed by QR1s. Two genes, putatively encoding a QR1 and a QR2, respectively, were isolated from an expressed sequence tag collection derived from the epidermis of a diploid wheat Triticum monococcum L. 24 h after inoculation with the powdery mildew fungus Blumeria graminis (DC) EO Speer f. sp. tritici Em. Marchal. Northern analysis and tissue-specific RT-PCR showed that TmQR1 was repressed while TmQR2 was induced in the epidermis during powdery mildew infection. Heterologous expression of TmQR2 in Escherichia coli confirmed that the gene encoded a functional, dicumarol-inhibitable QR2 that could use either NADH or NADPH as an electron donor. The localization of dicumarol-inhibitable QR2 activity around powdery mildew infection sites was accomplished using a histochemical technique, based on tetrazolium dye reduction.     

Purification and crystallization of rat liver NAD(P)H:(quinone-acceptor) oxidoreductase by cibacron blue affinity chromatography: identification of a new and potent inhibitor

Cytosolic NAD(P)H:(quinone-acceptor) oxidoreductase (EC 1.6.99.2) is a widely distributed, FAD-containing enzyme that catalyzes the obligatory two-electron reduction of quinones. Cibacron Blue is an inhibitor of this enzyme comparable in potency to dicoumarol. Pure quinone reductase was obtained from the livers of Sudan II (1-[2,4-dimethylphenylazo]-2-naphthol)-treated rats in a single step by Cibacron Blue-agarose chromatography. Cibacron Blue is a competitive inhibitor with respect to NADH (Ki = 170 nM) and is a noncompetitive inhibitor with respect to menadione (Ki = 540 nM). Addition of Cibacron Blue to quinone reductase resulted in a decrease and red shift of the enzyme-bound FAD peak at 450 nm. The titration of the absorbance changes for both FAD and Cibacron Blue could be fitted to curves describing an equilibrium binding equation with a KD of 300 nM and one binding site per enzyme subunit. Furthermore, the Cibacron Blue difference spectrum that resulted from binding to quinone reductase was abolished by dicoumarol. Significant amino acid homology between quinone reductase and the nucleotide binding regions of enzymes that bind to Cibacron Blue was found. These data indicate that Cibacron Blue is a useful ligand for the purification of quinone reductase and a new probe for its NAD(P)H binding site. Conditions for crystallizing rat liver quinone reductase are also described.     

Cellular knock-down of quinone reductase 2: a laborious road to successful inhibition by RNA interference

NRH:quinone oxidoreductase 2 (QR2) is a long forgotten oxidoreductive enzyme that metabolizes quinones and binds melatonin. We used the potency of the RNA interference (RNAi)-mediated gene silencing to build a cellular model in which the role of QR2 could be studied. Because standard approaches were poorly successful, we successively used: (1) two chemically synthesized fluorescent small interfering RNA (siRNA) duplexes designed and tested for their gene silencing capacity leading to a maximal 40% QR2 gene silencing 48h post-transfection; (2) double transfection and cell-sorting of high fluorescent siRNA-transfected HT22 cells further enhancing QR2 RNAi silencing to 88%; (3) stable QR2 knock-down HT22 cell lines established with H1and U6 promoter driven QR2 short hairpin RNA (shRNA) encoding vectors, resulting in a 71-80% reduction of QR2 enzymatic activity in both QR2 shRNA HT22 cells. Finally, as a first step in the study of this cellular model, we observed a 42-48% reduction of menadione/BNAH-mediated toxicity in QR2 shRNA cells compared to the wild-type HT22 cells. Although becoming widespread and in some cases effective, siRNA-mediated cellular knock-down proves in the present work to be of marginal efficiency. Much development is required for this technique to be of general application.     

X-ray diffraction analyses of crystals of rat liver NAD(P)H:(quinone-acceptor) oxidoreductase containing cibacron blue

NAD(P)H:(quinone-acceptor) oxidoreductase (EC 1.6.99.2) is a widely distributed dicoumarol-inhibitable FAD-containing protein that catalyzes the obligatory two-electron reduction of quinones. The enzyme plays an important role in protecting animal cells against quinone toxicity and may be involved in the vitamin K-dependent blood coagulation cascade. Cocrystallization of rat liver quinone reductase with Cibacron blue, a potent inhibitor with respect to NAD(P)H, was achieved by the method of vapor diffusion in the presence of ammonium sulfate and low concentrations of polyethylene glycol. X-ray diffraction analysis showed these blue crystalline platelets to be monoclinic and to belong to the space group P2(1) (a = 71.6 A, b = 107.1 A, c = 87.8 A and beta = 92.60 degrees) with two dimers in the asymmetric unit. The crystals diffract to a resolution of at least 2.8 A.     

An NADH:Quinone Oxidoreductase Active during Biodegradation by the Brown-Rot Basidiomycete Gloeophyllum trabeum

The brown-rot basidiomycete Gloeophyllum trabeum uses a quinone redox cycle to generate extracellular Fenton reagent, a key component of the biodegradative system expressed by this highly destructive wood decay fungus. The hitherto uncharacterized quinone reductase that drives this cycle is a potential target for inhibitors of wood decay. We have identified the major quinone reductase expressed by G. trabeum under conditions that elicit high levels of quinone redox cycling. The enzyme comprises two identical 22-kDa subunits, each with one molecule of flavin mononucleotide. It is specific for NADH as the reductant and uses the quinones produced by G. trabeum (2,5-dimethoxy-1,4-benzoquinone and 4,5-dimethoxy-1,2-benzoquinone) as electron acceptors. The affinity of the reductase for these quinones is so high that precise kinetic parameters were not obtainable, but it is clear that k_cat/K_m for the quinones is greater than 10⁸ M⁻¹ s⁻¹. The reductase is encoded by a gene with substantial similarity to NAD(P)H:quinone reductase genes from other fungi. The G. trabeum quinone reductase may function in quinone detoxification, a role often proposed for these enzymes, but we hypothesize that the fungus has recruited it to drive extracellular oxyradical production.     

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

Abstract

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.     

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.