The reaction of menadione with haemoglobin. Mechanism and effect of superoxide dismutase.

1. Menadione was found to react with both the haem groups and the beta-93 thiol groups of haemoglobin. 2. It oxidized the haem groups of oxyhaemoglobin, giving mainly methaemoglobin and a smaller amount of haemichrome. The reaction rate was decrease in the presence of catalase and markedly accelerated in the presence of superoxide dismutase. It is proposed that the overall reaction involves the initial reversible formation of methaemoglobin and the semiquinone, and that the effect of superoxide dismutase is to prevent the reverse reaction, by removing superoxide and hene O2-. E.s.r. evidence for the information of the semi-quinone and its reactions is presented. 3. The reaction of menadione with the beta-93 thiol groups of haemoglobin appeared to be similar to that with other thiols, forming the 3-thioether derivative of menadione, but it was also accompanied by reduction of methaemoglobin. This reduction was prevented by superoxide dismutase, but appeared to be caused by the semiquinone radical, which was produced as an intermediate. 4. Reduced glutathione functioned only to a limited extent as a scavenger of the menadione semiquinone. Its main reaction was directly with menadione to form the thioether. Ascorbate was a more efficient scavenger, and accelerated the oxidation of oxyhaemoglobin by menadione. 5. The significance of these findings in relation to menadione-induced erythrocyte haemolysis is discussed.     

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.     

Changes in the membrane ATPase activity of erythrocytes and Candida guilliermondii under the action of roseofungin

Changes in the Mg-ATPase and Na, K-ATPase activity of the rat erythrocyte and Candida guilliermondii membranes under the effect of roseofungin were studied. The antibiotic was totally bound to the isolated plasmatic membranes of Candida guilliermondii, up to 3 micrograms of the antibiotic per 1 microgram of the yeast protein. The Mg-APTase activity of these membranes was slightly inhibited by the antibiotic. The activity of Na, K-ATPase was almost completely inhibited even at 0.04 mg of roseofungin per 1 mg of protein. Much higher concentrations of the antibiotic inhibited the Mg-ATPase and Na, K-ATPase activity of the erythrocyte membranes to a less extent.     

Cooperative binding of primycin and gramicidin on erythrocyte membranes. A cation transport study

In this paper the authors present a comparative study of the actions of the antibiotics primycin and gramicidin on the erythrocyte membrane permeability. It has been found that both antibiotics have a nonlinear effect on the membrane permeability. Above a threshold antibiotic concentration, which is characteristic of the type of the antibiotic, the cation permeability of the erythrocyte membranes increases sharply. In the range of nonlinearity the transport-kinetic curves level off before achieving the equilibrium radioactive ion distribution between the extra- and intracellular spaces. A stochastic model of the cooperative and aspecific incorporation of antibiotic molecules into the membrane explains the experimental findings. The authors conclude that membrane permeability increases at the places where two or more antibiotic molecules form aggregates in the membrane.     

Intramolecular electron transfer in proteins. Radiolysis study of the reductive activation of daunorubicin complexed in egg white apo-riboflavin binding protein

Daunorubicin, an anthracycline antitumor antibiotic, can be complexed in egg white apo-riboflavin binding protein. The reduction of this complex was studied by gamma-radiolysis and pulse radiolysis using COO.- free radicals as reductants. The final products are 7-deoxydaunomycinone intercalated in the protein and thiol groups coming from the reduction of disulfide bonds of the protein, in the respective proportions of 90% and 10%. One-electron reduction of the complex gives daunorubicin semiquinone radical and a disulfide anion. The rate constants of the reactions of COO.- ions with the complex and with the disulfide bond in the protein alone are respectively equal to 2.4 x 10(8) mol-1.L.s-1 and 6.4 x 10(7) mol-1.L.s-1. Daunorubicin semiquinone decays by a first-order process, the rate constant of which is independent of the initial protein and radical concentrations. Without protein, daunorubicin semiquinone undergoes a disproportionation-comproportionation equilibrium [Houée-Levin, C., Gardès-Albert, M., Ferradini, C., Faraggi, M., & Klapper, M. (1985) FEBS Lett. 179, 46-50]. We propose that a protein residue reduces semiquinone by an intramolecular path. This creates an electron hole in the protein which may alter its function. This reduction process is very different from the reduction mechanism of riboflavin binding protein by the same reductant [Faraggi, M., Steiner, J.P., & Klapper, M.H. (1985) Biochemistry 24, 3273-3279]. These results suggest a new deleterious pathway to explain the antitumor and/or cytotoxic effect of this drug.     

Generation of daunomycin radicals on the outer side of the erythrocyte membrane

The generation of the daunomycin semiquinone was studied in intact red blood cells under CO atmosphere by ESR spectroscopy. The undialyzed hemolysates and the spin broadening agent chromium oxalate quenched the ESR signal, suggesting external location of the ESR-detectable radicals and their slow diffusion inside. A constant outward flow of O2- was detected by monitoring the approach to the steady state of the ESR signal of Cu,Zn superoxide dismutase externally added to red blood cells plus daunomycin in air. This suggests a reductase on the outer side of the erythrocyte membrane as the source of daunomycin radicals.     

Intramolecular semiquinone disproportionation in DNA. Pulse radiolysis study of the one-electron reduction of daunorubicin intercalated in DNA

The one-electron reduction of daunorubicin, a quinonic antitumor antibiotic, intercalated in DNA was studied by pulse radiolysis using carboxyl radicals as reductants. The reaction's first stage is the daunorubicin semiquinone formation (k = 1.9 x 10(8) mol-1.dm3.s-1) in a way entirely consistent with a simple competition between .COO- disproportionation and the drug reduction. The semiquinone drug disappears by a first-order reaction (k = 1340 s-1) producing the hydroquinone form. This reaction leads to an equilibrium similar to the one without DNA and the equilibrium constant is very close to its value free in water (Kc approximately 25). In addition, the stoichiometry of the first-order reaction is the one of a dismutation process. Therefore, it appears that the disproportionation occurs along an intramolecular path across DNA. This migration takes place under our experimental conditions, over a distance of ca. 100 base pairs, with a mobility of ca. 4.4 X 10(-11) m2.V-1.s-1, similar in magnitude to an excess electron mobility in doped organic polymers.     

The effect of metal ions on the electrochemistry of the antitumor antibiotic streptonigrin

The effect of transition metal ions on the electrochemistry of 6-methoxy-5,8-quinolinedione (L1), 7-amino-6-methoxy-5,8-quinolinedione (L2) and the antitumor antibiotic streptonigrin (SN) was studied. In 10% methanol/water, the one-electron reduction of quinones L1 and L2 to the corresponding semiquinones is shifted to more positive potentials upon addition of one equivalent of Zn(II), Ni(II), Co(II) or Cd(II) and is consistent with formation of a 1:1 complex involving the quinone(N) and adjacent quinone(O). Similar results are observed for Cu(II) and Mn(II), but the redox chemistry is also complicated by metal-based redox chemistry. The addition of further equivalents of M(II) results in a number of different coordination and electrochemical processes including formation of 1:1 and 2:1 complexes of the quinone, semiquinone and dianion. Under similar conditions, the 1:1 SN 2,2'-bipyridyl metal complex undergoes a reversible one-electron reduction to the semiquinone. The redox potential of the quinone in SN was shifted positive in the presence of the metal ions, but both the magnitude of the shift, and the relative influence of the metals was different to ligands L1 and L2. The changes in redox chemistry of SN compared with L1 and L2 are consistent with the formation of the 2,2-bipyridyl complexes in which there is weaker coordination to the quinone(O) in ring A of SN. These results suggest that in vivo, metal ions such as Zn(II), Cu(II) and Mn(II) facilitate the initial reduction of streptonigrin to the semiquinone by capturing the semiquinone after SN is reduced by biological reductants.     

One-electron reduction of hepatic NADH-cytochrome b5 reductase as studied by pulse radiolysis

The reduction of flavin in hepatic NADH-cytochrome b5 reductase by the hydrated electron (eaq-) was investigated by pulse radiolysis. The eaq- reduced the flavin of NADH-cytochrome b5 reductase to form the red semiquinone between pH 5 and 9. The spectrum of the red semiquinone differs from that of enzyme reduced by dithionite in the presence of NAD+. After the first phase of the reduction, conversion of the red to blue semiquinone was observed at acidic pH. Resulting products are the blue (neutral) or red (anionic) semiquinone or a mixture of the two forms. The pK value for this flavin radical was approximately 6.3. Subsequently, the semiquinone form reacted by dismutation to form the oxidized and the fully reduced forms of the enzyme with a rate constant of 1 x 10(3) M-1 s-1 at pH 7.1. In the presence of NAD+, eaq- reacted with NAD+ to yield NAD(.). Subsequently, NAD. transferred an electron to NAD+-bound oxidized enzyme to form the blue and red semiquinone or mixture of the two forms of the enzyme, where pK value of this flavin radical was approximately 6.3. The blue semiquinone obtained at acidic pH was found to convert to the red semiquinone with a first order rate constant of 90 s-1, where the rates were not affected by pH or the concentration of NAD+. The final product is NAD+-bound red semiquinone of the enzyme.     

Vitamin K-dependent carboxylase: Evidence for a semiquinone radical intermediate

Nanosecond laser flash photolysis has been used to produce and identify the vitamin K semiquinone (radical) from vitamin K dihydroquinone and to observe its formation and decay in the presence of vitamin K-dependent carboxylase (epoxidase). The activity of vitamin K-dependent carboxylase is not decreased by exposure to the laser. Absorbance of the semiquinone is proportional to enzyme concentration and is stimulated by a synthetic substrate, PheLeuGluGluIle. Stabilization of the semiquinone is observed in the presence of the enzyme. The semiquinone is rapidly destroyed in the presence of inhibitors of vitamin K-dependent carboxylase and vitamin K epoxidase.     

Inhibition of anthracycline semiquinone formation by ICRF-187 (Dexrazoxane) in cells

The formation of semiquinone free radicals of doxorubicin, epirubicin, daunorubicin, and idarubicin was measured by electron paramagnetic resonance (EPR) spectroscopy in hypoxic suspensions of chinese hamster ovary (CHO) cells. The amount of semiquinone produced was in the order idarubicin > doxorubicin > daunorubicin > epirubicin. The idarubicin semiquinone signal was both the fastest to be formed and to decay. Idarubicin, which was the most lipophilic of the anthracyclines studied, also displayed the fastest fluorescence-measured cellular uptake of drug. Thus, it was concluded that semiquinone formation was dependent upon the rate of cellular uptake. Lysed cell suspensions were also shown to be capable of producing the doxorubicin semiquinone in the presence of added NADPH. The cardioprotective agent dexrazoxane (ICRF-187) was observed to decrease the amount of doxorubicin semiquinone observed in cell suspensions. Dexrazoxane also decreased the amount of doxorubicin semiquinone observed in the NADPH-lysed cell suspension mixture. Neither bipyridine nor deferoxamine decreased NADPH-dependent doxorubicin semiquinone formation. These results suggest that dexrazoxane does not decrease doxorubicin semiquinone formation through an iron complex formed from hydrolyzed dexrazoxane. Dexrazoxane may be inhibiting an NADPH-dependent enzyme.     

Kinetics of reduction of Clostridium pasteurianum rubredoxin by laser photoreduced spinach ferredoxin:NADP+ reductase and free flavins. Electron transfer within a protein-protein complex

The kinetics of reduction of oxidized Clostridium pasteurianum rubredoxin (Rdox) by free flavin semiquinones generated by the laser flash photolysis technique and by spinach ferredoxin:NADP+ reductase (FNR) semiquinone (also produced by flavin semiquinone reduction) have been investigated under anaerobic conditions. 5-Deazariboflavin semiquinone (5-dRf) rapidly reduces oxidized rubredoxin (Rdox) (k = 3.0 X 10(8) M-1 S-1) and oxidized ferredoxin:NADP+ reductase (FNRox) to the semiquinone level (k = 5.5 X 10(8) M-1 S-1). Lumiflavin semiquinone reduces Rdox more slowly (k = 1.3 X 10(7) M-1 S-1) and is not measurably reactive with FNRox. Absorption difference spectroscopy and difference CD indicate that Rdox and FNRox form a 1:1 complex at low ionic strength (10 mM), which is completely dissociated at higher ionic strength (310 mM). Apparent second order rate constants for reduction of Rdox in its free and complexed state by lumiflavin semiquinone are the same. Reduction of Rdox (both free and complexed) by free FNR semiquinone and intracomplex electron transfer were investigated using 5-dRf as the reductant. At I = 10 mM, a first order rate constant of 2.0 X 10(3) S-1 was obtained, which corresponds to the processes involved in intracomplex electron transfer from FNR semiquinone to Rdox. A second order reaction between free FNR semiquinone and complexed Rdox was also observed to occur (k = 5 X 10(7) M-1 S-1). At I = 310 mM, these reactions are not observed and the reaction of FNR semiquinone with free Rdox is second order (k = 4 X 10(6) M-1 S-1).     

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.     

Kinetic model of the operation of a 2-electron switch in the photosynthetic reaction center of bacteria

A mathematical model, describing the binary oscillation of the concentration of semiquinone form of the secondary acceptor (ubiquinone) in photosynthetic reaction center of purple bacteria is proposed. This model takes into account both the changes of the ubiquinone state when the chromatophores are subjected to short flashes of light, and the successive dark relaxation of the semiquinone form. The model allows to calculate such characteristics as the dependence of the flash number, the stationary level of semiquinone form which is being established, when the flash number increases, the velocity which the concentration of semiquinone form is aspirating towards this stationary level and other characteristics. The model shows that the quantum yield of primary charge separation on the reaction center is higher after odd-number flashes then after even-number flashes.     

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.     

High-stability semiquinone intermediate in nitrate reductase A (NarGHI) from Escherichia coli is located in a quinol oxidation site close to heme bD

Quinol/nitrate oxidoreductase (NarGHI) is the first enzyme involved in respiratory denitrification in prokaryotes. Although this complex in E. coli is known to operate with both ubi and menaquinones, the location and the number of quinol binding sites remain elusive. NarGHI strongly stabilizes a semiquinone radical located within the dihemic anchor subunit NarI. To identify its location and function, we used a combination of mutagenesis, kinetics, EPR, and ENDOR spectroscopies. For the NarGHIH66Y and NarGHIH187Y mutants lacking the distal heme bD, no EPR signal of the semiquinone was observed. In contrast, a semiquinone was detected in the NarGHIH56Y mutant lacking the proximal heme bP. Its thermodynamic properties and spectroscopic characteristics, as revealed by Q-band EPR and ENDOR spectroscopies, are identical to those observed in the native enzyme. The substitution by Ala of the Lys86 residue close to heme bD, which was previously proposed to be in a quinol oxidation site of NarGHI (QD), also leads to the loss of the EPR signal of the semiquinone, although both hemes are present. Enzymatic assays carried out on the NarGHIK86A mutant reveal that the substitution dramatically reduces the rate of oxidation of both mena and ubiquinol analogues. These observations demonstrate that the semiquinone observed in NarI is strongly associated with heme bD and that Lys86 is required for its stabilization. Overall, our results indicate that the semiquinone is located within the quinol oxidation site QD. Details of the possible binding motif of the semiquinone and mechanistic implications are discussed.     

Semiquinone and ascorbyl radicals in the gut fluids of caterpillars measured with EPR spectrometry

The biological activity of phenolic compounds ingested by caterpillars is commonly believed to result from their oxidation, although the products of oxidation have been well-characterized in only a few cases. The initial oxidation products of phenols (semiquinone or phenoxyl radicals) can be measured with electron paramagnetic resonance (EPR) spectrometry. In this study semiquinone radicals formed from tannic acid and gallic acid in the gut fluids of two species of caterpillars were measured. In Orgyia leucostigma, in which ingested phenols are not oxidized, semiquinone radicals were absent or at very low intensities. By contrast, in Malacosoma disstria, in which ingested phenols are oxidized, high semiquinone radical intensities were measured. In the absence of detectable levels of semiquinone radicals, ascorbyl radicals were detected in the EPR spectra instead. High molar ratios of ascorbate to phenols in an artificial diet produced ascorbyl radicals in the midgut fluids of both species, while diets containing low molar ratios produced semiquinone radicals. Similar results were obtained in M. disstria fed the leaves of red oak or sugar maple. The results of this study provide further evidence that ascorbate is an essential antioxidant that prevents the oxidation of phenols in the gut fluids of caterpillars, and demonstrate that EPR spectrometry is a valuable method for determining the degree of oxidative activation of phenols ingested by herbivorous insects.     

The unusual redox properties of flavocytochrome P450 BM3 flavodoxin domain

Flavocytochrome P450 BM3 FMN domain is unique among the family of flavodoxins and homologues, in not forming a stable neutral blue FMN semiquinone radical. Anaerobic, one-electron reduction of the isolated domain over the pH 7-9.5 range showed that it forms an anionic red semiquinone that disproportionates slowly (0.014s(-1) at pH 7). The rate of disproportionation decreased at higher pH, indicating that protonation of the anionic semiquinone is an important feature of the mechanism. The reduction potential for the oxidised-semiquinone couple was determined to be -240mV and was largely independent of pH. The semiquinone appears, therefore, to be kinetically trapped by a slow protonation event, enabling it to act as a low-potential electron donor to the P450 heme.     

Spin-trapping and direct electron spin resonance investigations of the redox metabolism of quinone anticancer drugs

The superoxide free radical has been spin trapped in microsomal incubations containing adriamycin, daunorubicin, and mitomycin C. The time sequence of the appearance of the spin-trapped superoxide and the semiquinone radical metabolite of these quinone-containing anticancer drugs indicates that air oxidation of the semiquinone is responsible for the superoxide formation. Superoxide dismutase prevents the formation of the superoxide spin adducts. Microsomal incubations containing anthracyclines intercalated in DNA produce much less superoxide than incubations free of DNA. The first unambiguous ESR evidence for the semiquinone metabolite of mitomycin C in a biological system is also presented.     

Primary and secondary electron transfer in hexane-solubilized proteolipid complexes of Rhodopseudomonas sphaeroides R-26

Primary electron transfer in hexane-solubilized reaction center proteolipid complexes is similar to that in detergent-solubilized reaction centers or chromatophores when diaminodurene is electron donor. Approximate values for the extinction coefficients of ubisemiquinone and the diaminodurene cation can be calculated. The primary and secondary quinone sites are dissimilar and results in only the transient formation of a semiquinone anion pair after two photochemical turnovers. One of the semiquinone anions decays rapidly, the remaining one and the diaminodurene cation have long lifetimes. Disproportionation between the semiquinone anions does not occur.