Sigmatropic reactions of the aziridinyl semiquinone species. Why aziridinyl benzoquinones are metabolically more stable than aziridinyl indoloquinones

Described herein is the chemistry of aziridinyl semiquinone species, which are formed upon one-electron metabolic reduction of aziridinyl quinone antitumor agents. The semiquinone species undergo a type of electrocyclic reaction known as a 1,5-sigmatropic shift of hydrogen. This reaction converts the aziridinyl group to both ethylamino and amino groups resulting in a loss of cytotoxicity. Since the radical anion conjugate base does not undergo ring opening as fast as the semiquinone, it was possible to determine the semiquinone pK(a) values by plotting the percent sigmatropic products versus pH. Aziridinyl quinones based on benzoquinones, such as DZQ and AZQ, possess semiquinone pK(a) values below neutrality. In contrast, an indole-based aziridinyl quinone possesses a semiquinone pK(a) value of 9.3. Single electron reduction of DZQ and AZQ by NADPH: cytochrome P-450 reductase at physiological pH therefore affords the radical anion without any sigmatropic rearrangement products. In contrast, the same reduction of an aziridinyl indoloquinone affords the semiquinone which is rapidly converted to sigmatropic rearrangement products. These findings suggest that aziridinyl quinone antitumor agents based on indoles will be rapidly inactivated by one electron-reductive metabolism. A noteworthy example is the indoloquinone agent EO9, which is rapidly metabolized in vivo. In contrast, benzoquinone-based aziridinyl quinone antitumor agents such as AZQ, DZQ, and the new benzoquinone analogue RH1 do not suffer from this problem.     

Hydroxyl radical production by free and DNA-bound aminoquinone antibiotics and its role in DNA degradation. Electron spin resonance detection of hydroxyl radicals by spin trapping.

The reduced antitumor antibiotic mitomycin C in aqueous solution exposed to air gives a 36-line electron spin resonance spectrum of the semiquinone identified by computer simulation. Incubation of this radical with the spin trap N-tert-butyl-alpha-phenylnitrone (PBN) gives the PBN.OH nitroxide radical identified by independent generation. This nitroxide radical is also formed from similar treatment of a DNA to which mitomycin C is covalently attached. Incubation of the semiquinone from mitomycin C, mitomycin B, or streptonigrin (SN) with catalase or with superoxide dismutase inhibits the generation of OH, implying the intermediacy of H2O2 and O2 in its formation. The formation of the spin-trapped nitroxide radical is similarly inhibited by EDTA, suggesting the intermediacy of trace metal ions in the generation of hydroxyl radicals from SN. The results are consistent with the generation by the aminoquinone antibiotics in vivo of OH. already implicated in the degradation of DNA.     

The function of photosystem I. Quantum chemical insight into the role of tryptophan-quinone interactions

Quantum chemical calculations have provided evidence for the role of tryptophan residues in the electron transfer process of photosystem I (PS-I). The interaction of Trp with quinone acceptors and their radical anions in the A(1) site of PS-I has been modeled by various indole-quinone and indole-semiquinone complexes. MP2 optimizations show that, while neutral quinones and an indole molecule prefer a pi-stacked arrangement, semiquinone radical anions prefer a T-stacked conformation with significant N-H...pi hydrogen bonding interactions. Comparison of density functional calculations of electronic g-tensors with electron paramagnetic resonance data strongly suggests that hydrogen-bonded T-shaped arrangements occur upon reduction of quinone acceptors without an extended side chain (e.g., duroquinone or naphthoquinone), when reconstituted into the phylloquinone-depleted A(1) site of PS-I. In contrast, for the native phylloquinone (vitamin K(1), Q(K)), reorientation of the semiquinone radical anion is prevented by side chain-protein interactions. For a fixed pi-stacked arrangement, the extent of the intermolecular interaction is reduced upon one-electron reduction. This corresponds to a lowering of the redox potential of the P(700)(+)*Q(K)(-)* radical pair, due to interactions of Q(K) with a tryptophan. Together with the comparably weak hydrogen bonding in PS-I, the proposed model explains the very negative redox potential of the A(1) site, needed for forward electron transfer. T-stacking hydrogen bonds to semiquinones may also have to be considered in many other electron transfer processes in living organisms.     

The effects of pH and semiquinone formation on the oxidation-reduction potentials of flavin mononucleotide. A reappraisal.

Calculation shows that there is poor agreement between frequently cited values for the midpoint redox potentials of the two one-electron steps in the reduction of flavin mononucleotide and equations for the lines that relate these potentials to pH and that use the published pKa values for the three redox states of the flavin [Draper, R. & Ingraham, L.L. (1969) Arch. Biochem. Biophys. 125, 802-808]. Equilibrium data for the first step in the reduction obtained by pulse radiolysis [Anderson, R.F. (1983) Biochim. Biophys. Acta 722, 158-162] show much closer agreement with theory and lead to values for the semiquinone formation constant of flavin mononucleotide that are close to those derived from measurements of the radical concentration using ESR spectroscopy. It is concluded that the data from the second method are more reliable. The redox potentials for flavin mononucleotide at pH 7.0 and 20 degrees C are calculated to be -0.207 V for the overall two-electron reduction (Em), -0.313 V for reduction of the oxidized flavin to the semiquinone (E2) and -0.101 V for the reduction of the semiquinone to the hydroquinone (E1). Information is provided to allow calculation of the three redox potentials at other pH values in the physiological range.     

Genotoxicity of streptonigrin: a review

Streptonigrin (SN, CAS no. 3930-19-6) is an aminoquinone antitumor antibiotic isolated from cultures of Streptomyces flocculus. This compound is a member of a group of antitumor agents which possess the aminoquinone moiety and that includes also mitomycin C, porfiromycin, actinomycin, rifamycin and geldanamycin. Because of the potential use of SN in clinical chemotherapy, the study of its genotoxicity has considerable practical significance.SN inhibits the synthesis of DNA and RNA, causes DNA strand breaks after reduction with NADH, induces unscheduled DNA synthesis and DNA adducts and inhibits topoisomerase II. At the chromosome level, this antibiotic causes chromosome damage and increases the frequency of sister-chromatid exchanges.SN cleaves DNA in cell-free systems by a mechanism that involves complexing with metal ions and autoxidation of the quinone moiety to semiquinone in the presence of NADH with production of oxygen-derived reactive species. Recent evidence strongly suggests that the clastogenic action of this compound is partially mediated by free radicals. The present review aims at summarizing past and current knowledge concerning the genotoxic effects of SN.     

High-resolution two-dimensional 1H and 14N hyperfine sublevel correlation spectroscopy of the primary quinone of photosystem II

Quinones are naturally occurring isoprenoids that are widely exploited by photosynthetic reaction centers. Protein interactions modify the properties of quinones such that similar quinone species can perform diverse functions in reaction centers. Both type I and type II (oxygenic and nonoxygenic, respectively) reaction centers contain quinone cofactors that serve very different functions as the redox potential of similar quinones can operate at up to 800 mV lower reduction potential when present in type I reaction centers. However, the factors that determine quinone function in energy transduction remain unclear. It is thought that the location of the quinone cofactor, the geometry of its binding site, and the "smart" matrix effects from the surrounding protein environment greatly influence the functional properties of quinones. Photosystem II offers a unique system for the investigation of the factors that influence quinone function in energy transduction. It contains identical plastoquinones in the primary and secondary quinone acceptor sites, Q(A) and Q(B), which exhibit very different functional properties. This study is focused on elucidating the tuning and control of the primary semiquinone state, Q(A)(-), of photosystem II. We utilize high-resolution two-dimensional hyperfine sublevel correlation spectroscopy to directly probe the strength and orientation of the hydrogen bonds of the Q(A)(-) state with the surrounding protein environment of photosystem II. We observe two asymmetric hydrogen bonding interactions of reduced Q(A)(-) in which the strength of each hydrogen bond is affected by the relative nonplanarity of the bond. This study confirms the importance of hydrogen bonds in the redox tuning of the primary semiquinone state of photosystem II.     

The metabolism of menadione (2-methyl-1,4-naphthoquinone) by isolated hepatocytes. A study of the implications of oxidative stress in intact cells

The cytotoxic effects of many quinones are thought to be mediated through their one-electron reduction to semiquinone radicals, which subsequently enter redox cycles with molecular oxygen to produce active oxygen species and oxidative stress. The two-electron reduction of quinones to diols, mediated by DT-diaphorase (NAD(P)H: (quinone-acceptor) oxidoreductase), may therefore represent a detoxifying pathway which protects the cell from the formation of these reactive intermediates. By using menadione (2-methyl-1,4-naphthoquinone) and isolated hepatocytes, the relative contribution of the two pathways to quinone metabolism has been studied and a protective role for DT-diaphorase demonstrated. Moreover, in the presence of cytotoxic concentrations of menadione rapid changes in intracellular thiol and Ca2+ homeostasis were observed. These were associated with alterations in the surface structure of the hepatocytes which may be an early indication of cytotoxicity.     

An electron spin resonance study of the reduction of peroxides by anthracycline semiquinones

Direct and spin-trapping electron spin resonance methods have been used to study the reactivity of semiquinone radicals from the anthracycline antibiotics daunorubicin and adriamycin towards peroxides (hydrogen peroxide, t-butyl hydroperoxide and cumene hydroperoxide). Semiquinone radicals were generated by one-electron reduction of anthracyclines, using xanthine/xanthine oxidase. It is shown that the semiquinones are effective reducing agents for all the peroxides. From spin-trapping experiments it is inferred that the radical product is either OH (from H2O2) or an alkoxyl radical (from the hydroperoxides) which undergoes beta-scission to give the methyl radical. The rate constant for reaction of semiquinone with H2O2 is estimated to be approx. 10(4)-10(5) M-1 X s-1. The reduction does not appear to require catalysis by metal ions.     

Electrochemical study of the complexes of aspartame with Cu(II), Ni(II) and Zn(II) ions in the aqueous medium

The voltammetric behaviours of aspartame in the presence of some metal ions (Cu(II), Ni(II), Zn(II)) were investigated. In the presence of aspartame, copper ions reduced at two stages with quasi-reversible one-electron and, with increasing the aspartame (L) concentration, Cu(II)L(2) complex reduces at one-stage with irreversible two-electron reaction (-0.322 V). Zn(II)-aspartame complex (logbeta=3.70) was recognized by a cathodic peak at -1.320 V. Ni(II)-aspartame complex (logbeta=6.52) is reduced at the more positive potential (-0.87 V) than that of the hydrated Ni(II) ions (-1.088 V). In the case of the reduction of Ni(II) ions, aspartame serves as a catalyst. From electronic spectra data of the complexes, their stoichiometries of 1:2 (metal-ligand) in aqueous medium are determined. The greatness of these logarithmic values is agreement with Irwing-Williams series (NiZn).     

The reductive activation of the antitumor drug RH1 to its semiquinone free radical by NADPH cytochrome P450 reductase and by HCT116 human colon cancer cells

RH1 (2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone), which is currently in clinical trials, is a diaziridinyl benzoquinone bioreductive anticancer drug that was designed to be activated by the obligate two-electron reductive enzyme NAD(P)H quinone oxidoreductase 1 (NQO1). In this electron paramagnetic resonance (EPR) study we showed that RH1 was reductively activated by the one-electron reductive enzyme NADPH cytochrome P450 reductase and by a suspension of HCT116 human colon cancer cells to yield a semiquinone free radical. As shown by EPR spin trapping experiments RH1 was reductively activated by cytochrome P450 reductase and underwent redox cycling to produce damaging hydroxyl radicals in reactions that were both H₂O₂- and iron-dependent. Thus, reductive activation by cytochrome P450 reductase or other reductases to produce a semiquinone that can redox cycle to produce damaging hydroxyl radicals and/or DNA-reactive alkylating species may contribute to the potent cell growth inhibitory effects of RH1. These results also suggest that selection of patients for treatment with RH1 based on their expression levels of NQO1 may be problematic.     

Steady-state and laser flash induced photoreduction of yeast glutathione reductase by 5-deazariboflavin and by a viologen analogue: stabilization of flavin adenine dinucleotide semiquinone species by complexation

Steady-state and laser flash photolysis techniques have been used to examine the photoreduction of yeast glutathione reductase by the one-electron reduction products of 5-deazariboflavin and the viologen analogue 1,1'-propylene-2,2'-bipyridyl. Steady-state photoreduction of the enzyme with the viologen generates the two-electron-reduced form, whereas photoreduction with deazaflavin generates the anion semiquinone. Flash photolysis indicates that the product of viologen radical reduction is also a semiquinone, suggesting that this species is rapidly further reduced by viologen in the steady-state experiment to form the EH2 enzyme. This reduction is apparently inhibited when deazaflavin is the photoreductant, perhaps due to complexation of the anion semiquinone with deazaflavin. Steady-state experiments demonstrate that complexation of the anion semiquinone with NADP+ also inhibits further reduction. Both one-electron reduction reactions of oxidized glutathione reductase proceed at close to diffusion-controlled rates (second-order rate constants = 10(8)-10(9) M-1 s-1), despite the relatively buried nature of the FAD cofactor. Addition of NADP+ and oxidized glutathione produced no effects on the kinetics of the initial entry of the electron into the enzyme. No kinetic evidence of intramolecular electron transfer involving the FAD and the protein disulfide was obtained during or subsequent to the initial one-electron reduction process. Thus, if this reaction occurs in the semiquinone, it must be quite rapid (k greater than 8000 s-1).     

Temporary decrease in renal quinone reductase activity induced by chronic administration of estradiol to male Syrian hamsters. Increased superoxide formation by redox cycling of estrogen

Cytochrome P-450-mediated redox cycling between the synthetic estrogen diethylstilbestrol (DES) and diethylstilbestrol-4',4"-quinone (DES Q) has previously been demonstrated. Cytochrome P-450 reductase catalyzes the reduction of DES Q presumably via a semiquinone formed by one-electron reduction. A reducing action of NAD(P)H quinone reductase (EC 1.6.99.2) mediating two-electron reduction of DES Q has been investigated in the present work. Quinone reductase catalyzed the conversion in the presence of NADH or NADPH of DES Q to 53-65% Z-DES, a marker product of reduction. Dicumarol (15 microM), a known specific inhibitor of quinone reductase, inhibited this reduction almost completely. Using microsomes from Syrian hamster kidney, a target organ of estrogen-induced carcinogenesis, the reduction of DES Q was only partially inhibited by dicumarol. Apparent Km values of quinone reductase and cytochrome P-450 reductase were 17.25 and 11.9 microM, respectively. These data demonstrate that in hamster kidney, quinone reductase and cytochrome P-450 reductase compete for the reduction of DES Q. Microsomal 02-. radical generation was stimulated 10-fold over base levels by the addition of 100 microM DES Q. The formation of 02-. radicals was inhibited by addition of superoxide dismutase (0.2 mg/ml) or by 2'-AMP or NADP, known inhibitors of cytochrome P-450 reductase. In contrast, dicumarol enhanced microsome-mediated 02-. formation. It is concluded that cytochrome P-450 reductase in hamster kidney microsomes mediates one-electron reduction of estrogen quinones to free radicals (semiquinones), which may subsequently enter redox cycling with molecular oxygen to form 02-.. Moreover, quinone reductase reduces DES Q directly to E- and Z-DES, and thus may prevent the formation of toxic intermediates during redox cycling of estrogens. Measurements of quinone reductase activity in liver and kidney of hamsters treated with estrogen for various lengths of time revealed a temporary decrease in activity by 80% specifically in the kidney after 1 month of chronic treatment with estradiol. Thus, a temporary decrease in quinone reductase activity, which occurred specifically in estrogen-exposed hamster kidney, may enhance the formation of free radical intermediates generated during biotransformation of estrogens.     

Source of superoxide anion radical in aerobic mixtures consisting of NAD[P]H, 5-methylphenazinium methyl sulfate and nitroblue tetrazolium chloride

The source of superoxide anion radical (O2-.) in aerobic mixtures consisting of NAD[P]H, 5-methylphenazinium methyl sulfate (or its 1-methoxy derivative) and tetrazolium salt was investigated using superoxide dismutase (SOD), Mn(II), ferricytochrome-C, and epinephrine as probes. NAD[P]H + phenazine + O2 was found to reduce nitroblue tetrazolium, iodonitrotetrazolium, and thiazolyl blue in a manner sensitive to agents that dismutase O2-., viz., SOD and Mn(II). It also mediated the reduction of ferricytochrome-C, and augmented the autooxidation of epinephrine to the adrenochrome, without a tetrazolium salt present in the medium. The autooxidation of epinephrine, but not the reduction of ferricytochrome-C, was found to be sensitive to SOD. Nitroblue tetrazolium, either singly or in combination with SOD, did not stimulate the reduction of ferricytochrome-C. The oxidation of NADH, mediated by a catalytically low concentration of phenazine(+O2), was augmented two-fold by SOD. These observations are consistent with, and lend support to, a scheme of redox events (Scheme-3) wherein it is proposed that the source of O2-. in the NAD[P]H + phenazine + tetrazolium(+O2) system is the reduced phenazine, that the tetrazoinyl radical (a one-electron reduction product of tetrazolium) may not reduce O2 to O2-., that the redox reaction between semiquinone radicals of phenazine and O2 is reversible, and that the disproportionation of semiquinone radicals constitutes an important rate-limiting reaction in the expression of phenazine redox couple.     

Calmodulin activates intramolecular electron transfer between the two flavins of neuronal nitric oxide synthase flavin domain

The neuronal NO synthase (nNOS) flavin domain, which has similar redox properties to those of NADPH-cytochrome P450 reductase (P450R), contains binding sites for calmodulin, FAD, FMN, and NADPH. The aim of this study is to elucidate the mechanism of activation of the flavin domain by calcium/calmodulin (Ca(2+)/CaM). In this study, we used the recombinant nNOS flavin domains, which include or delete the calmodulin (CaM)-binding site. The air-stable semiquinone of the nNOS flavin domains showed similar redox properties to the corresponding FAD-FMNH(&z.ccirf;) of P450R. In the absence or presence of Ca(2+)/CaM, the rates of reduction of an FAD-FMN pair by NADPH have been investigated at different wavelengths, 457, 504 and 590 nm by using a stopped-flow technique and a rapid scan spectrophotometry. The reduction of the oxidized enzyme (FAD-FMN) by NADPH proceeds by both one-electron equivalent and two-electron equivalent mechanisms, and the formation of semiquinone (increase of absorbance at 590 nm) was significantly increased in the presence of Ca(2+)/CaM. The air-stable semiquinone form of the enzyme was also rapidly reduced by NADPH. The results suggest that an intramolecular one-electron transfer between the two flavins is activated by the binding of Ca(2+)/CaM. The F(1)H(2), which is the fully reduced form of the air-stable semiquinone, can donate one electron to the electron acceptor, cytochrome c. The proposed mechanism of activation by Ca(2+)/CaM complex is discussed on the basis of that provided by P450R.     

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.     

An NADH:quinone oxidoreductase of the halotolerant bacterium Ba1 is specifically dependent on sodium ions

The rate of NADH oxidation by inverted membrane vesicles prepared from the halotolerant bacterium Ba1 of the Dead Sea is increased specifically by sodium ions, as observed earlier in whole cells. The site of this sodium effect is identified as the NADH: quinone oxidoreductase, similarly to the other such system known, Vibrio alginolyticus (H. Tokuda and T. Unemoto (1984) J. Biol. Chem. 259, 7785-7790). Sodium accelerates quinone reduction severalfold, but oxidation of the quinol, with oxygen as terminal electron acceptor, is unaffected. The sodium-dependent pathway of quinone reduction exhibits higher apparent affinity to extraneous quinone (Q-2) than the sodium-insensitive pathway, and is specifically inhibited by 2-heptyl-4-hydroxyquinoline N-oxide. ESR spectra of the membranes contain a feature at g = 1.98 which is tentatively identified as one originating from semiquinone. This feature is increased by NADH and decreased by addition of Na+, suggesting that, as proposed from different kinds of evidence for the V. alginolyticus system, sodium affects the semiquinone reduction step. As in the other system, the site of sodium stimulation in Ba1 probably corresponds to the site of sodium translocation, which was shown earlier (S. Ken-Dror, R. Shnaiderman, and Y. Avi-Dor (1984) Arch. Biochem. Biophys. 229, 640-649) to be linked directly to a redox reaction in the respiratory chain.     

The reductive activation of the antitumor drug RH1 to its semiquinone free radical by NADPH cytochrome P450 reductase and by HCT116 human colon cancer cells

RH1 (2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone), which is currently in clinical trials, is a diaziridinyl benzoquinone bioreductive anticancer drug that was designed to be activated by the obligate two-electron reductive enzyme NAD(P)H quinone oxidoreductase 1 (NQO1). In this electron paramagnetic resonance (EPR) study we showed that RH1 was reductively activated by the one-electron reductive enzyme NADPH cytochrome P450 reductase and by a suspension of HCT116 human colon cancer cells to yield a semiquinone free radical. As shown by EPR spin trapping experiments RH1 was reductively activated by cytochrome P450 reductase and underwent redox cycling to produce damaging hydroxyl radicals in reactions that were both H₂O₂- and iron-dependent. Thus, reductive activation by cytochrome P450 reductase or other reductases to produce a semiquinone that can redox cycle to produce damaging hydroxyl radicals and/or DNA-reactive alkylating species may contribute to the potent cell growth inhibitory effects of RH1. These results also suggest that selection of patients for treatment with RH1 based on their expression levels of NQO1 may be problematic.     

Oxidation of DOPAC by nitric oxide: effect of superoxide dismutase

This study aimed to characterize the redox interaction between 3,4-dihydroxyphenylacetic acid (DOPAC) and nitric oxide (.NO), and to assess the reductive and oxidative decay pathways of the DOPAC semiquinone originating from this interaction. The reaction between DOPAC and.NO led to the formation of the DOPAC semiquinone radical, detected by electron paramagnetic resonance (EPR) and stabilized by Mg(2+), and the nitrosyl anion detected as nitrosylmyoglobin. The EPR signal corresponding to the DOPAC semiquinone was modulated as follows: (i) it was suppressed by glutathione and ascorbic acid with the formation of new EPR spectra corresponding to the glutathionyl and ascorbyl radical, respectively; (ii) it was enhanced by Cu,Zn-superoxide dismutase; the enzyme also accelerated the decay of the semiquinone species to DOPAC quinone. These results are interpreted as a one-electron oxidation of DOPAC by.NO; the reductive decay of the semiquinone back to DOPAC was facilitated by reducing agents, such as glutathione and ascorbate, whereas the oxidative decay to DOPAC quinone was facilitated by superoxide dismutase. The latter effect is understood in terms of a reversible conversion of nitrosyl anion to.NO by the enzyme. The biological relevance of these reactions is also discussed in terms of the reactivity of peroxynitrite towards DOPAC as a model with implications for aerobic conditions.     

Thermodynamic properties of the semiquinone and its binding site in the ubiquinol-cytochrome c (c2) oxidoreductase of respiratory and photosynthetic systems

The antimycin-sensitive ubisemiquinone radical (QC) of the ubiquinol-cytochrome c oxidoreductase of submitochondrial particles and chromatophores of Rhodopseudomonas sphaeroides Ga has been studied by a combination of redox potentiometry and EPR spectroscopy. This g = 2.005 radical signal appears at physiological pH values and increases in intensity with increasing pH up to pH 7.6 in submitochondrial particles and pH 9.0 in R. sphaeroides after which its intensity remains unchanged. The Em7 (ubiquinone/quinol) of the signal, estimated from redox titration data is 80 mV for submitochondrial particles, and 150 mV in chromatophores. Each of these values is higher than that of the quinone pool by 20 mV in submitochondrial particles and 60 mV in R. sphaeroides. This indicates that the quinone at the binding site is out of equilibrium with the pool, and that binding site preferentially binds quinol over quinone. Analysis of the shapes of the semiquinone titration curves, taken together with the midpoint elevation, indicates a quinone-binding site: cytochrome c1 stoichiometry of 1:1 in both submitochondrial particles and chromatophores. At its maximal intensity, the semiquinone concentration at the binding site is 0.26 in submitochondrial particles (greater than pH 7.6) and 0.4 in chromatophores (greater than pH 9.0). In both systems, the midpoint of the ubiquinone/ubisemiquinone couple is constant as the pH is raised up to the pH of maximal semiquinone formation whereafter it becomes more negative at the rate of -60 mV/pH unit. The midpoint of the ubisemiquinone/quinol couple, on the other hand, varies by -120 mV/pH unit at pH values up to the transition pH, after which it, too, changes by -60 mV/pH unit. This seemingly anomalous behavior may be explained by invoking a protonated group at or near the quinone-binding site whose pK corresponds to the pH transition point in the quinone/semiquinone/quinol redox chemistry when the site is free or when quinone or quinol occupies the site. This pK is elevated to at least pH 9.0 in submitochondrial particles and 10.5 in R. sphaeroides when semiquinone is bound to the site.     

Hepatic low-level chemiluminescence during redox cycling of menadione and the menadione-glutathione conjugate: relation to glutathione and NAD(P)H:quinone reductase (DT-diaphorase) activity

Formation of excited species such as singlet molecular oxygen during redox cycling (one-electron reduction-oxidation) was detected by low-level chemiluminescence emitted from perfused rat liver and isolated hepatocytes supplemented with the quinone, menadione (vitamin K3). Chemiluminescence was augmented when the two-electron reduction of the quinone catalyzed by NAD(P)H:quinone reductase was inhibited by dicoumarol, thus underlining the protective function of this enzyme also known as DT-diaphorase. Interference with NADPH supply by inhibition of energy-linked transhydrogenase by rhein or of mitochondrial electron transfer by antimycin A led to a depression in the level of photoemission. Unexpectedly, glutathione depletion of the liver led to a lowering of chemiluminescence elicited by menadione, whereas conversely the depletion of glutathione led to increased chemiluminescence levels when a hydroperoxide was added instead of the quinone. As the GSH conjugate of menadione, 2-methyl-3-glutathionyl-1,4-naphthoquinone, studied with microsomes, was shown also to be capable of redox cycling, we conclude that menadione-induced chemiluminescence of the perfused rat liver does not only arise from menadione itself but from the menadione-GSH conjugate as well. Therefore, the conjugation of the quinone with glutathione is not in itself of protective nature and does not abolish semiquinone formation. A biologically useful aspect of conjugate formation resides in the facilitation of biliary elimination from the liver. Nonenzymatic formation of the conjugate from menadione and GSH in vitro was found to be accompanied by the formation of aggressive oxygen species.