One-electron Reduction Of Quinones Research Articles

In cellulo monitoring of quinone reductase activity and reactive oxygen species production during the redox cycling of 1,2 and 1,4 quinones

Quinones are highly reactive molecules that readily undergo either one- or two-electron reduction. One-electron reduction of quinones or their derivatives by enzymes such as cytochrome P450 reductase or other flavoproteins generates unstable semiquinones, which undergo redox cycling in the presence of molecular oxygen leading to the formation of highly reactive oxygen species. Quinone reductases 1 and 2 (QR1 and QR2) catalyze the two-electron reduction of quinones to form hydroquinones, which can be removed from the cell by conjugation of the hydroxyl with glucuronide or sulfate thus avoiding its autoxidation and the formation of free radicals and highly reactive oxygen species. This characteristic confers a detoxifying enzyme role to QR1 and QR2, even if this character is strongly linked to the excretion capacity of the cell. Using EPR spectroscopy and confocal microscopy we demonstrated that the amount of reactive oxygen species (ROS) produced by Chinese hamster ovary (CHO) cells overexpressing QR1 or QR2 compared to naive CHO cells was determined by the quinone structural type. Indeed, whereas the amount of ROS produced in the cell was strongly decreased with para-quinones such as menadione in the presence of quinone reductase 1 or 2, a strong increase in ROS was recorded with ortho-quinones such as adrenochrome, aminochrome, dopachrome, or 3,5-di-tert-butyl-o-benzoquinone in cells overexpressing QR, especially QR2. These differences could originate from the excretion process, which is different for para- and ortho-quinones. These results are of particular interest in the case of dopamine considering the association of QR2 with various neurological disorders such as Parkinson disease.

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 ( K m app ) was ∼0.3 nM and the apparent maximum velocity ( V max app ) was 16 U mg −1. Substrate inhibition was observed above 5 nM. NADH was the electron donor, K m app = 340 μ M and V max app = 46 U mg - 1 . FAD was also a cofactor with a K m app of 3.1 μM and V max 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.

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

Quinones including antitumor quinones undergo facile reduction and oxidation. One-electron reduction of a quinone gives the semiquinone radicals while two-electron reduction gives the hydroquinonel. In 1969-70, Iyanagi and Yamazaki2,3 reported the mechanism of quinone reduction by flavin enzymes, and the reduction of quinones and oxygen by flavin enzymes falls into three mechanistic categories: one-electron, two-electron and mixed-type reactions. NAD(P)H:quinone oxidoreductase(QR), also known as DT-diaphorase contains one FAD as prosthetic group, catalyzes the obligatory two-electron reduction of quinone to hydroquinone. On the other hand, microsomal NADPHcytochrome P450 reductase (P450 reductase) and NADH-cytochome b5 reductase, and mitochondrial NADH-ubiquinone oxidoreductase, and ferredoxin: NADP+ reductase catalyze typical one electron reduction of bivalent quinones. Xanthine oxidase/ dehydrogenase catalyzes both reactions of one-electron and two-electron. These results have contributed greatly to the study on the formation of free-radicals in biological systems, especially in the quinone-mediated cyotoxicity.

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 (Ca 2+/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 Ca 2+/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 Ca 2+/CaM.

Kinetics of redox interaction between substituted quinones and ascorbate under aerobic conditions

One-electron reduction of quinones (Q) by ascorbate (AscH −); (1) AscH −+Q→Q − +Asc − +H +, followed by the oxidation of semiquinone (Q − ) by molecular oxygen; (2) Q − +O 2→Q+O 2 − , results in the catalytic oxidation of ascorbate (with Q as a catalyst) and formation of active forms of oxygen. Along with enzymatic redox cycling of Q, this process may be related to Q cytotoxicity and underlie an antitumor activity of some Qs. In this work, the kinetics of oxygen consumption accompanied the interaction of ascorbate with 55 Qs including substituted 1,4- and 1,2-benzoquinones, naphthoquinones and other quinoid compounds were studied in 50 mM sodium phosphate buffer, pH 7.40, at 37°C by using the Clark electrode technique. The capability of Q to catalyze ascorbate oxidation was characterized by the effective value of k EFF calculated from the initial rate of oxygen consumption ( R OX) by the equation R OX= k EFF[Q][AscH −] as well as by a temporary change in R OX. The correlation of k EFF with one-electron reduction potential, E(Q/Q − ), showed a sigma-like plot, the same for different kinds of Qs. Only the Qs which reduction potential E(Q/Q − ) ranged from nearly −250 to +50 mV displayed a pronounced catalytic activity, k EFF increased with shifting E(Q/Q − ) to positive values. The following linear correlation between k EFF (in M −1 s −1) and E(Q/Q − ) (in mV) might be suggested for these Qs: lg( k EFF)=3.91+0.0143 E(Q/Q − ). In contrast, Qs with E(Q/Q − )<−250 mV and E(Q/Q − )>+50 mV showed no measurable catalytic activity. The Qs studied displayed a wide variety in the kinetic regularities of oxygen consumption. When E(Q/Q − ) was more negative than −100 mV, Q displayed a simple (‘standard’) kinetic behavior— R OX was proportional to [AscH −][Q] independently of concentration of individual reagents, [AscH −] and [Q]; R OX did not decrease with time if [AscH −] was held constant; Q recycling was almost reversible. Meanwhile, Qs with E(Q/Q − )>−100 mV demonstrated a dramatic deviation from the ‘standard’ behavior that was manifested by the fast decrease in R OX with time and non-linear dependence of even starting values of R OX on [Q] and [AscH −]. These deviations were caused basically by the participation of Q − in side reactions different from (2). The above findings were confirmed by kinetic computer simulations. Some biological implications of Q–AscH − interaction were discussed.

Genetic evidence for coenzyme Q requirement in plasma membrane electron transport.

Plasma membranes isolated from wild-type Saccharomyces cerevisiae crude membrane fractions catalyzed NADH oxidation using a variety of electron acceptors, such as ferricyanide, cytochrome c, and ascorbate free radical. Plasma membranes from the deletion mutant strain coq3delta, defective in coenzyme Q (ubiquinone) biosynthesis, were completely devoid of coenzyme Q6 and contained greatly diminished levels of NADH-ascorbate free radical reductase activity (about 10% of wild-type yeasts). In contrast, the lack of coenzyme Q6 in these membranes resulted in only a partial inhibition of either the ferricyanide or cytochrome-c reductase. Coenzyme Q dependence of ferricyanide and cytochrome-c reductases was based mainly on superoxide generation by one-electron reduction of quinones to semiquinones. Ascorbate free radical reductase was unique because it was highly dependent on coenzyme Q and did not involve superoxide since it was not affected by superoxide dismutase (SOD). Both coenzyme Q6 and NADH-ascorbate free radical reductase were rescued in plasma membranes derived from a strain obtained by transformation of the coq3delta strain with a single-copy plasmid bearing the wild type COQ3 gene and in plasma membranes isolated form the coq3delta strain grown in the presence of coenzyme Q6. The enzyme activity was inhibited by the quinone antagonists chloroquine and dicumarol, and after membrane solubilization with the nondenaturing detergent Zwittergent 3-14. The various inhibitors used did not affect residual ascorbate free radical reductase of the coq3delta strain. Ascorbate free radical reductase was not altered significantly in mutants atp2delta and cor1delta which are also respiration-deficient but not defective in ubiquinone biosynthesis, demonstrating that the lack of ascorbate free radical reductase in coq3delta mutants is related solely to the inability to synthesize ubiquinone and not to the respiratory-defective phenotype. For the first time, our results provide genetic evidence for the participation of ubiquinone in NADH-ascorbate free radical reductase, as a source of electrons for transmembrane ascorbate stabilization.

Calculated One-Electron Reduction Potentials and Solvation Structures for Selected p-Benzoquinones in Water

The one-electron reduction of quinones is important not only in electrochemistry but also in biochemical energy storage, energy utilization, and organic chemcial reactions. Thermodynamic cycles are investigated to estimate aqueous one-electron reduction potentials for the redox indicators p-benzoquinone and p-duroquinone, as well as chloro-substituted p-benzoquinones. Gas-phase reduction free energy differences are approximated from electron affinities calculated by using the hybrid Hartree−Fock/density-functional B3LYP method, a semiempirical quantum chemical method that expresses a molecule's exchange-correlation energy as a weighted sum of Hartree−Fock, local, and gradient-corrected density-functional energies. Free energy perturbation theory was used with molecular dynamics simulations (at constant temperature, pressure, and number of atoms) to estimate hydration free energy differences. Calculated one-electron reduction potentials for the quinones are within 10−190 meV of experimental values. An exce...