YC-1-like potentiation of nitric oxide-dependent activation of soluble guanylate cyclase by adrenochrom

The influence of adrenochrome and YC-1 activation of human platelet soluble guanylate cyclase was investigated. Adrenochrome (0.1–10.0 μM) had no effect on the basal activity, but it potentiated in a concentration- dependent manner the spermine NONO-induced activation of this enzyme. Adrenochrome also sensitized guanylate towards nitric oxide (NO) and produced the leftward shift of the spermine NONO concentration response curve. Addition of adrenochrome decreased the YC-1-induced leftward shift of the spermine NONO concentration response curve. Adrenochrome also inhibited enzyme activation byYC-1. Thus, synergistic activation of NO-stimulated guanylate cyclase activity by adrenochrome represents a new biochemical effect of this compound and indicates that adrenochrome may act as an endogenous regulator of the NO-dependent stimulation of soluble guanylate cyclase. This new property of adrenochrome, similar to YC-1 but more effective, should be taken into consideration especially under conditions of adrenochrome overproduction in the body.     

Spectroscopic studies on the photoreaction of choline oxidase, a flavoprotein, with covalently bound flavin

Formation of the anionic flavosemiquinone was observed spectrophotometrically during the anaerobic photo-irradiation of Alcaligenes sp. choline oxidase in the presence of EDTA. Further irradiation slowly converted the semiquinone form into the fully reduced state. The presence of a catalytic amount of riboflavin greatly enhances the photoreduction rate not only to the semiquinone state but also to the fully reduced state. This semiquinone species has low reactivity toward the substrate, choline or betaine aldehyde, as well as toward oxygen. This low reactivity toward oxygen is unique to the semiquinone form of a flavoprotein oxidase. The oxidized enzyme forms a complex with betaine, the product of the enzymatic reaction of choline oxidase. The dissociation constant for this complex was found to be 17 mM by spectroscopic titration. Anaerobic photo-irradiation of the enzyme with a saturating amount of betaine in the absence of EDTA produces, with no detectable semiquinone formation, an absorption spectrum which resembles (but significantly differs from) that of the fully reduced form. This species was found to comprise two flavin species. One of them is rapidly oxidized to the oxidized form by oxygen and is thus assigned as the fully reduced state. The other is converted slowly to the oxidized form upon aerobic standing in the dark. We tentatively assigned this latter species as a C(4a)-adduct. Formaldehyde was detected as a product of this photoreaction. The amount of formaldehyde formed coincided with that of the fully reduced enzyme. On the basis of the results obtained we propose a mechanism of the photoreaction of the enzyme in the presence of betaine where a C(4a)-adduct and the fully reduced enzyme via an N(5)-adduct are formed. Betaine also affects the dithionite reduction. In the dithionite reduction of the oxidized enzyme, the semiquinone species is an intermediate in the conversion of the oxidized to the fully reduced form, while the reduction of the oxidized enzyme-betaine complex with dithionite produces the fully reduced form without any significant formation of the semiquinone species.     

Production of superoxide anions and hydrogen peroxide in Ehrlich ascites tumour cell nuclei.

Nuclei isolated from Ehrlich-Lettré ascites tumour cells catalyze the co-oxidation of epinephrine to adrenochrome in the presence of NADPH. Adrenochrome formation is sensitive to superoxide dismutase but not to scavengers of hydroxyl radicals or singlet oxygen. Addition of NADPH also initiates the production of hydrogen peroxide. Moreover measurements of superoxide dismutase activity indicate the presence of this enzyme in the ascites cell nuclei, although the sensitivity of adrenochrome formation to externally added superoxide dismutase indicates that the endogenous enzyme is not sufficient for a complete protection from superoxide radicals.     

Pulse radiolysis studies of antitumor quinones: Radical lifetimes,reactivity with oxygen,and one-electron reduction potentials

The formation of semiquinone free radicals from antitumor drugs has been studied by pulse radiolysis. The semiquinone free radicals are reactive and have short half-lives in aqueous media under anaerobic conditions. The half-lives of the radicals formed from adriamycin, mitomycin C, and 2,5-diaziridinyl-3,6-bis(carboethoxy)amine-1,4-benzoquinone (AZQ) are 50,100, and 200 μs, respectively. The mean diffusion distance of the semiquinone free radical is less than 0.6 μm. In the presence of molecular oxygen the half-life of the semiquinone free radical is shortened. Adriamycin semiquinone reacts rapidly with oxygen, k = 4.4 × 10⁷m^?1s^?1. In air-saturated buffer the half-life of adriamycin semiquinone radical can be calculated to be 8 μs with a mean diffusion distance of less than 0.1 μm. If the half-lives in buffer are comparable to those within a cell, semiquinone free radicals must be generated close to the site at which they produce a biological effect. One-electron reduction potentials (E₇¹) were determined and were AZQ, ?168 mV, adrenochrome, ?253 mV, mitomycin C, ?271 mV, adriamycin, ?292 mV, daunomycin, ?305 mV, and anthracenedione, ?348 mV. Enzymatic one-electron reduction of these antitumor quinones by NADPH-cytochrome P-450 reductase increased at more positive values of quinone E₇¹.     

Production of superoxide anions and hydrogen peroxide in Ehrlich ascites tumour cell nuclei

Nuclei isolated from Ehrlich-Lettré ascites tumour cells catalyze the co-oxidation of epinphrine to adrenochrome in the presence of NADPH. Adrenochrome formation is senstive to superoxide dismutase but not to scavengers of hydroxyl radicals or singler oxygen. Addition of NADPH also initiates the production of hydrogen peroxide. Moreover measurements of superoxide dismutase activity indicate the presence of this enzyme in the ascites cell nuclei, although the sensitivity of adrenochrome formation to externally added superoxide dismutase indicates that the endogenous enzyme is not sufficient for a complete protection from superoxide radicals.     

The epinephrine assay for superoxide: why dopamine does not work

Superoxide oxidizes epinephrine to a semiquinone, initiating a series of reactions leading to the colored product adrenochrome. This popular assay for superoxide is more sensitive at higher pH, and it does not work if dopamine is used instead of epinephrine. A kinetic analysis shows that these effects can be explained by competing reactions that lower the yield of the observed product. The catecholamine quinone may cyclize to form the absorbing product, or it may be reduced back to the semiquinone by superoxide. For epinephrine, the quinone cyclizes quickly and adrenochrome formation dominates, but for dopamine, the quinone cyclizes slowly and the back reaction prevails. The yield of adrenochrome increases if the epinephrine semiquinone reacts with O₂ to form more superoxide, but this reaction competes with disproportionation of the semiquinone. Because disproportionation slows as pH increases, both superoxide formation and the yield of adrenochrome increase at higher pH.     

Redox cycling of anthracyclines by cardiac mitochondria. I. Anthracycline radical formation by NADH dehydrogenase

In the present study we have used beef heart submitochondrial preparations (BH-SMP) to demonstrate that a component of mitochondrial Complex I, probably the NADH dehydrogenase flavin, is the mitochondrial site of anthracycline reduction. During forward electron transport, the anthracyclines doxorubicin (Adriamycin) and daunorubicin acted as one-electron acceptors for BH-SMP (i.e. were reduced to semiquinone radical species) only when NADH was used as substrate; succinate and ascorbate were without effect. Inhibitor experiments (rotenone, amytal, piericidin A) indicated that the anthracycline reduction site lies on the substrate side of ubiquinone. Doxorubicin and daunorubicin semiquinone radicals were readily detected by ESR spectroscopy. Doxorubicin and daunorubicin semiquinone radicals (g congruent to 2.004, signal width congruent to 4.5 G) reacted avidly with molecular oxygen, presumably to produce O2-, to complete the redox cycle. The identification of Complex I as the site of anthracycline reduction was confirmed by studies of ATP-energized reverse electron transport using succinate or ascorbate as substrates, in the presence of antimycin A or KCN respiratory blocks. Doxorubicin and daunorubicin inhibited the reduction of NAD+ to NADH during reverse electron transport. Furthermore, during reverse electron transport in the absence of added NAD+, doxorubicin and daunorubicin addition caused oxygen consumption due to reduction of molecular oxygen (to O2-) by the anthracycline semiquinone radicals. With succinate as electron source both thenoyltrifluoroacetone (an inhibitor of Complex II) and rotenone blocked oxygen consumption, but with ascorbate as electron source only rotenone was an effective inhibitor. NADH oxidation by doxorubicin during BH-SMP forward electron transport had a KM of 99 microM and a Vmax of 30 nmol X min-1 X mg-1 (at pH 7.4 and 23 degrees C); values for daunorubicin were 71 microM and 37 nmol X min-1 X mg-1. Oxygen consumption at pH 7.2 and 37 degrees C exhibited KM values of 65 microM for doxorubicin and 47 microM for daunorubicin, and Vmax values of 116 nmol X min-1 X mg-1 for doxorubicin and 114 nmol X min-1 X mg-1 for daunorubicin. In marked contrast with these results, 5-iminodaunodrubicin (a new anthracycline with diminished cardiotoxic potential) exhibited little or no tendency to undergo reduction, or to redox cycle with BH-SMP. Redox cycling of anthracyclines by mitochondrial NADH dehydrogenase is shown, in the accompanying paper (Doroshow, J. H., and Davies, K. J. A. (1986) J. Biol. Chem. 261, 3068-3074), to generate O2-, H2O2, and OH which may underlie the cardiotoxicity of these antitumor agents.     

Mechanism(s) for the metabolism of mitoxantrone: electron spin resonance and electrochemical studies

Mitoxantrone has been reported to lack certain properties that characterize quinone containing antitumor agents that undergo enzymatic reduction. These properties are the stimulation of NADPH oxidation, the stimulation of oxygen consumption by microsomes and reductases and, the absence of oxygen free radicals during these reactions. Having these properties implies the presence of a futile redox cycle that requires the generation and the oxidation of a semiquinone free radical. It would follow that if mitoxantrone does not redox cycle in the presence of reductases, then the semiquinone free radical is not produced or, if it is formed, it reacts quickly to form diamagnetic products. However, using liver microsomes, there are reports of the formation of the mitoxantrone free radial anion. In this paper we investigated the mitoxantrone free radical anion generated electrochemically and found that in the presence of oxygen it behaved like other semiquinones. That is, it is oxidized to the parent compound (presumably generating oxygen free radicals), indicating the ability to redox cycle. The reduction potential to generate such free radical in aqueous medium is very high (-0.79 V) when compared to diaziquone (-0.36 V) and Adriamycin (-0.6 V). This suggests that mitoxantrone may not be a substrate for reductases. Under reductive conditions with purified NADPH cytochrome P-450 reductase which very easily reduces diaziquone and Adriamycin, mitoxantrone was not reduced. However, under the same conditions, mitoxantrone was oxidized by the prototype oxidase horseradish peroxidase with the production of a mitoxantrone free radical. This oxidation was accompanied by a drastic change in color and the formation of a dark precipitate. Because microsomes contain a variety of enzymes, we suggest that the previously observed free radical in microsomes is probably due to the oxidation of mitoxantrone. In this theory, this product is probably a polymer which would not require oxygen to be formed. Thus, under oxidative conditions, the mitoxantrone free radical cation will also display impaired redox activity.     

Superoxide and hydrogen peroxide production and NADPH oxidation stimulated by nitrofurantoin in lung microsomes: possible implications for toxicity.

The addition of nitrofurantoin to aerobic incubation mixtures containing rat lung microsomes strongly enhanced the generation of adrenochrome from epinephrine. Adrenochrome formation in this system was blocked by superoxide dismutase, but not by catalase. Hydrogen peroxide production was also strongly enhanced by nitrofurantoin in these preparations; superoxide dismutase did not significantly alter the amount of H₂O₂ measured, but no H₂O₂ was detected in incubation mixtures in the presence of catalase. Nitrofurantoin enhanced the oxidation of NADPH in lung microsomal suspensions under aerobic conditions; the enhancement was unaffected by catalase but was partially prevented by superoxide dismutase. Neither adrenochrome formation nor H₂O₂ production were enhanced by nitrofurantoin under anaerobic (N₂) conditions, but NADPH oxidation in the presence of nitrofurantoin was greater under anaerobic conditions than under aerobic conditions. These results are consistent with the view that the redox cycling of nitrofurantoin in lung microsomes in the presence of oxygen results in the consumption of NADPH and the production of activated oxygen species, emphasizing some $\text{in vitro}$ metabolic similarities with the lung-toxic herbicide, paraquat.     

Spectral properties of 3-ketosteroid-delta 1-dehydrogenase from Nocardia corallina

3-Ketosteroid-delta 1-dehydrogenase from Nocardia corallina is a flavoenzyme that catalyzes 1,2-desaturation of 3-ketosteroid. The dehydrogenase generated complexes with 3-ketosteroids and phenolic steroids such as estradiol with remarkable perturbations of the visible spectrum. The enzyme did not make the adduct with sulfite ion, but could use molecular oxygen as the electron acceptor. The CD spectra of oxidized and steroid-bound enzymes exhibited positive dichroisms in the visible region which resembled those of flavoenzyme oxidases. The dehydrogenase led isosbestically to the stable red semiquinone species with large yields upon photochemical or dithionite reduction (at pH 7.4) in the presence of the steroid product, 1,4-androstadiene-3,17-dione, but in the absence of the steroid the yield of semiquinone was low and the fully reduced enzyme was obtained. Substrate titration also yielded the red flavo-semiquinone stoichiometrically and it was hard to generate the fully reduced form. The reduced enzyme was oxidized with molecular oxygen, but did not oxidize with ferricyanide. An EPR study of these half-reduced forms confirmed the presence of the radical species with the g = 2.004 signal. The dehydrogenase was rapidly reduced with an excess amount of 3-ketosteroid at about 80% yield at pH 7.4 under anaerobic conditions and the reduced species was altered to the stable red semiquinone species. The rate of this reaction was t1/2 = 28 min at pH 7.4, 130 min at pH 9.0 and 34 min at pH 6.4, respectively. These results indicate that the semiquinone species does not act directly in turnover of the dehydrogenase reaction. The results were compared with the spectral properties of general acyl-CoA dehydrogenases and acyl-CoA oxidase toward the mechanism of C1,2-dehydrogenation.     

Spin-Trapping and Direct Epr Investigations on the Hepatotoxic and Hepatocarcinogenic Actions of Luteoskyrin, An Anthraquinoid Mycotoxin Produced by Penicillium Islandicum Sopp. Generations of Superoxide Anion and Luteoskyrin Semiquinone Radical in the Redox Systems Consisted of Luteoskyrin and Liver Nadph- Or Nadh-Dependent Reductases

Luteoskyrin is a hepatotoxic and hepatocarcinogenic bisdihydroanthraquinone produced by Penicillium islandicum Sopp. By observing the EPR spectra of DMPO-spin adducts and luteoskyrin semiquinone radical, we investigated in vitro whether luteoskyrin is reduced to its semiquinone radical leading to the generation of active oxygen species in redox systems catalyzed by NADPH-dependent cytochrome reductases of the liver. We found (1) the formation of luteoskyrin semiquinone radical in the NADPH-cytochrome P-450 reductase system under anaerobic conditions, (2) the generation of O- in the systems composed of luteoskyrin, NAD(P)H, and either rat liver microsomal NADPH-cytochrome P-450 reductase or submitochondrial particles and (3) dicoumarol showed no effect on the O- generation in the case of submitochondrial particles. From these results we proposed that luteoskyrin liver injuries are induced by the active oxygen species generated in the process of autoxidation of luteoskyrin semiquinone radical which is produced in the one-electron redox systems catalyzed by the liver NAD(P)H-dependent cytochrome reductases.     

The involvement of oxygen radicals during the autoxidation of adrenalin.

1. In unbuffered alkaline solutions, autoxidizing adrenalin generates superoxide anions: both the scavenging by adrenalin itself, leading to adrenochrome, and the formation of nitrite from hydroxylamine are inhibited by superoxide dismutase. No hydroxyl radical could be detected. 2. The yield of hydrogen peroxide increases with pH in a way similar to that of adrenochrome and nitrite. The dissociated form of adrenalin (pK = 8.5) is proposed as the source of superoxide anions. 3. Superoxide dismutase delays rather than inhibits the reaction. In addition to the diminished formation of adrenochrome due to the scavenging of superoxide anions and re-reduction of the semiquinone by hydrogen peroxide, respectively, adrenochrome is further removed by hydrogen peroxide, with final products absorbing at 310 nm. 4. The diminished inhibitory effect of superoxide dismutase above pH 10 is due to superoxide-independent reactions. This effect is masked by the alkaline conversion of adrenochrome to indole compounds. 5. It is concluded that monitoring the absorption of adrenochrome in alkaline solutions does not produce reliable evidence for superoxide anions.     

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.     

Mechanism of aromatic hydroxylation. Properties of a model for pyridine nucleotide-dependent flavoprotein hydroxylases

A model (NADH-phenazine methosulfate-O₂) formally similar to pyridine nucleotide-dependent flavoprotein hydroxylases catalyzed the hydroxylation of several aromatic compounds. The hydroxylation was maximal at acid pH and was inhibited by ovine Superoxide dismutase, suggesting that perhydroxyl radicals might be intermediates in this process. The stoichiometry of the reaction indicated that a univalent reduction of oxygen was occurring. The correlation between the concentration of semiquinone and hydroxylation, and the inhibition of hydroxylation by ethanol which inhibited semiquinone oxidation, suggested the involvement of phenazine methosulfate-semiquinone. Activation of hydroxylation by Fe³⁺ and Cu²⁺ supported the contention that univalently reduced species of oxygen was involved in hydroxylation. Catalase was without effect on the hydroxylation by the model, ruling out H₂O₂ as an intermediate. A reaction sequence, involving a two-electron reduction of phenazine methosulfate to reduced phenazine methosulfate followed by disproportionation with phenazine methosulfate to generate the semiquinone, was proposed. The semiquinone could donate an electron to O₂ to generate O₂ which could be subsequently protonated to form the perhydroxyl radical.     

Redox cycling of adrenaline and adrenochrome catalysed by mitochondrial Complex I

Complex I in bovine heart submitochondrial particles catalyses the NADH-supported generation of superoxide anion; adrenaline is oxidised by superoxide to adrenochrome that, on its hand, is reduced by Complex I, thus establishing a redox cycle that amplifies the superoxide production. The routes in Complex I for superoxide formation and for adrenochrome reduction appear to be different, since they have a different sensitivity to Complex I inhibitors. The results are discussed in terms of current assays for superoxide detection and of pathologies linked to catecholamine oxidation.     

Properties of the radical intermediate obtained on oxidation of 2',7'-dichlorodihydrofluorescein, a probe for oxidative stress

Reduced "leuco" dyes such as dichlorodihydrofluorescein (DCFH(2)) are widely used as profluorescent probes for oxidative stress, although they require a catalyst to be oxidized by hydrogen peroxide and react indiscriminately with oxidizing radicals and the fluorescent product (DCF) is a potential photosensitizer of superoxide generation. In this study, key properties of the radical intermediate in oxidation ("semiquinone," DCFH(.-)/DCF(.)(-)) were measured, to help understand the reactions that can occur in biological systems. The intermediate was generated by oxidizing DCFH(2) or reducing DCF by radiolytically generated radicals and monitoring the reactions using kinetic spectrophotometry. The semiquinone showed pH-sensitive absorption spectral changes, decay kinetics (both in the absence and in the presence of oxygen), and reduction potential, all corresponding to prototropic dissociations with pK(a)'s of approximately 7.1 and 9.0. DCFH(2) has pK(a)'s in a similar region (8-9) and hence pH variations are potentially important in the use of this probe. The rate constant for reaction of the semiquinone with oxygen at pH 7.4 is 5.3 x 10(8) M(-1) s(-1): this reaction, rather than disproportionation of DCFH(.-)/DCF(.)(-), generates DCF in biological systems, concomitantly forming superoxide and hence H(2)O(2) to cycle the catalyst. The midpoint reduction potential of the couple DCF,H(+)/DCFH() is approximately -0.75 V vs. NHE at pH 7.4; DCF is unlikely to be reduced rapidly by common flavoprotein reductases.     

New sensitive agents for detecting singlet oxygen by electron spin resonance spectroscopy.

Free radicals are well-established transient intermediates in chemical and biological processes. Singlet oxygen, though not a free radical, is also a fairly common reactive chemical species. It is rare that singlet oxygen is studied with the electron spin resonance (ESR) technique in biological systems, because there are few suitable detecting agents. We have recently researched some semiquinone radicals. Specifically, our focus has been on bipyrazole derivatives, which slowly convert to semiquinone radicals in DMSO solution in the presence of potassium tert-butoxide and oxygen. These bipyrazole derivatives are dimers of 3-methyl-1-phenyl-2-pyrazolin-5-one and have anti-ischemic activities and free radical scavenging properties. In this work, we synthesized a new bipyrazole derivative, 4,4'-bis(1p-carboxyphenyl-3-methyl-5-hydroxyl)-pyrazole, DRD156. The resulting semiquinone radical, formed by reaction with singlet oxygen, was characterized by ESR spectroscopy. DRD156 gave no ESR signals from hydroxyl radical, superoxide, and hydrogen peroxide. DRD156, though, gives an ESR response with hypochlorite. This agent, nevertheless, has a much higher ability to detect singlet oxygen than traditional agents with the ESR technique.     

Structural and biochemical evidence for an enzymatic quinone redox cycle in Escherichia coli: identification of a novel quinol monooxygenase

Naturally synthesized quinones perform a variety of important cellular functions. Escherichia coli produce both ubiquinone and menaquinone, which are involved in electron transport. However, semiquinone intermediates produced during the one-electron reduction of these compounds, as well as through auto-oxidation of the hydroxyquinone product, generate reactive oxygen species that stress the cell. Here, we present the crystal structure of YgiN, a protein of hitherto unknown function. The three-dimensional fold of YgiN is similar to that of ActVA-Orf6 monooxygenase, which acts on hydroxyquinone substrates. YgiN shares a promoter with "modulator of drug activity B," a protein with activity similar to that of mammalian DT-diaphorase capable of reducing mendione. YgiN was able to reoxidize menadiol, the product of the "modulator of drug activity B" (MdaB) enzymatic reaction. We therefore refer to YgiN as quinol monooxygenase. Modulator of drug activity B is reported to be involved in the protection of cells from reactive oxygen species formed during single electron oxidation and reduction reactions. The enzymatic activities, together with the structural characterization of YgiN, lend evidence to the possible existence of a novel quinone redox cycle in E. coli.     

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.     

The tiron free radical as a sensitive indicator of chloroplastic photoautoxidation.

Wheat chloroplasts photochemically reduced molecular oxygen, as a Hill oxidant in the Mehler reaction, to superoxide anion which then oxidized added 1,2-dihydroxybenzene-3,5-disulfonate to its semiquinone, a comparatively stable free radical at pH 7. The last mentioned reaction was rapid in aqueous solution, but the rate of formation of 1,2-dihydroxybenzene-3,5-disulfonate semiquinone by the chloroplast system was calculated as T1 of 0.6 s. The Mehler reaction, or more specifically the univalent reduction of oxygen by Photosystem I, was rate-limiting so that the 1,2-dihydroxybenzene-3,5-disulfonate seniquinone was a useful spin probe for superoxide anion production at room temperature. The ESR signal of 1,2-dihydroxybenzene-3,5-disulfonate semiquinone was proportional to its steady state concentration and decayed in the dark with a T1/2 of 5-6 s. This oxygen-dependent signal was enhanced by mediation of chloroplastic oxygen reduction through methyl viologen. The superoxide anion scavengers ascorbate and L-epinephrine competitively obscured 1,2-dihydroxybenzene-3,5-disulfonate semiquinone formation, butadded superoxide dismutase was not as effective in this role. Partial inhibition by superoxide dismutase was achieved only by preincubation of Photosystem I enriched particles with ten times the endogenous concentration of superoxide dismutase. This and the persistence of a small amount of a 1,2-dihydroxybenzene-3,5-disulfonate (Tiron) oxidizing species in the dark supports the concept of Tiron accessibility but not the superoxide dismutase accessibility of superoxide anion bound in its formative enzyme complex. Benzoquinone and naphthoquinone disulfonate also reacted with superoxide anion, and supported both the Hill reaction and the Mehler reaction as final oxidants of both water and superoxide anion.