Beef Heart Submitochondrial Particles Research Articles

Isolation, Biological Activity Evaluation, Structure Elucidation, and Total Synthesis of Eliamid: A Novel Complex I Inhibitor

Eliamid is a secondary metabolite isolated from two bacterial strains. This molecule features a linear polyketide backbone terminated by a tetramic acid amide moiety. Among other biological activities, eliamid shows a high and specific cytostatic action on human lymphoma and cervix carcinoma cell lines. The 2,4-anti relative configuration of the C-2,C-4-dimethyl substituted amide fragment was assigned by means of Breit's rule. The absolute configuration of all stereocenters was determined by a combination of degradation methods, structural similarity analysis and total synthesis. The stereogenic centers were introduced by vinylogous Mukaiyama aldol reaction and two consecutive Myers alkylations. The use of pentafluorophenyl ester as acylation agent allowed the efficient formation of tetramic acid amide. The longest linear sequence in the synthesis consist of 13 steps and proceeds with 12% overall yield. Differential spectroscopy experiments with beef heart submitochondrial particles established that eliamid is a potent inhibitor of the NADH-ubiquinone oxidoreductase complex. Additionally, biosynthesis of eliamid was investigated by feeding experiments with (13)C-labeled precursors.

Iromycins from Streptomyces sp. and from synthesis: New inhibitors of the mitochondrial electron transport chain

Two new α-pyridone metabolites, iromycins E and F, were isolated from cultures of strain Streptomyces sp. Dra 17, thus expanding the recently discovered iromycin family. The inhibitory potential on the mitochondrial respiratory chain was examined and revealed that iromycin metabolites block NADH oxidation in beef heart submitochondrial particles with different efficacy, yet remarkably show only very low cytotoxicity. Difference spectroscopic studies indicated that iromycins inhibit the electron transport at the site of complex I (NADH-ubiquinone oxidoreductase). Derivatives of the natural products were semisynthetically prepared and provided detailed insights into structure–activity relationships. Drawn from these results, there are strong similarities with the piericidins, which are among the most potent complex I inhibitors of the mitochondrial electron transport chain. Furthermore, total synthesis afforded new analogues, and the non-natural iromycin S (IC 50 = 58 ng/mL) emerged as the most active compound, thus opening avenues of future studies with the iromycins as new valuable biochemical tools.

Novel Inhibitors of the Mitochondrial Respiratory Chain: Oximes and Pyrrolines Isolated fromPenicillium brevicompactumand Synthetic Analogues

The capacity of inhibition of the mammalian mitochondrial respiratory chain of brevioxime 5a, a natural insecticide compound isolated from Penicillium brevicompactum culture broth, and another 15 analogue compounds, other oximes 5b and 5c; two diastereomeric pyrrolidines 1c' and 1c' '; five pyrrolines 3c', 3c' ' (diastereomers between them), 3a, 3b, and 6; two oxazines 4c' and 4c' ' (also diastereomers between them); and four pyrrol derivatives 7-10, are analyzed in this paper. Compounds 3b, 3c', 3c' ', 4c', 4c' ', 5b, 5c, 6, and 10 were found to be inhibitors of the integrated electron transfer chain (NADH oxidase activity) in beef heart submitochondrial particles (SMP), establishing that all of them except compound 3b and 6 only affected to complex I of the mitochondrial respiratory chain. The most potent product was 5b, with an IC50 of 0.27 microM, similar to the IC50 values of other known complex I inhibitors. The diastereomeric pairs 1c'/1c' ', 3c'/3c' ', 4c'/4c' ', and 5c have not been previously described. Chemical characterization, on the basis of spectral data, is also shown.

Inhibition of the mitochondrial ATP synthesis by polygodial, a naturally occurring dialdehyde unsaturated sesquiterpene

Polygodial is a naturally occurring sesquiterpene dialdehyde that exhibits several pharmacologically interesting activities. Among them, its antifungal properties have been more thoroughly studied. The mitochondrial ATPase has been suggested as one of the possible targets for polygodial action. However, its mechanism of action is not well defined yet. The effect of polygodial on the mitochondrial energy metabolism is described in this paper. Polygodial inhibited ATP synthesis coupled to succinate oxidation in beef-heart submitochondrial particles at concentrations (IC 50 = 2.4 ± 0.1 μM) which marginally affected electron transport and ATPase activity (IC 50 = 97 ± 4 μM). A transitory stimulation of the electron transport in intact rat liver mitochondria in state 4 was also obtained at low polygodial concentrations (EC 50 = 20 ± 4 μM). These results suggest that polygodial uncouples ATP synthesis from electron transport at low concentrations. Similar concentrations of polygodial partially abolished the ANS fluorescence enhancement (IC 50 = 2.2 ± 0.4 μM) induced by succinate oxidation in submitochondrial particles but did not collapse the ΔpH. We postulate that polygodial uncouples mitochondrial ATP synthesis by affecting the electrical properties of the membrane surface and consequently collapsing the membrane potential (Δ ψ) and/or the localized transmembrane pH difference (ΔpH S) without affecting the ΔpH between the two bulk aqueous phases (ΔpH B). The relevance of these findings for the understanding of the biochemical basis of the antifungal activity of polygodial and the evaluation of its potentiality as a therapeutic agent are discussed.

Using cytochome c to monitor electron transport and inhibition in beef heart submitochondrial particles*

We present a two-part undergraduate laboratory exercise. In the first part, electron transport in bovine heart submitochondrial particles causing reduction of cytochrome c is monitored at 550 nm. Redox-active dyes have historically been used in most previous undergraduate laboratory exercises of this sort but do not demonstrate respiratory inhibition by antimycin A and rotenone. By using cytochrome c instead of redox-active dyes, it is possible to observe inhibition of electron transport in the presence of the aforementioned respiratory inhibitors. In the second part, students are asked to design a soluble redox chain between NADH and cytochrome c using catalytic amounts of redox-active dyes. The students are also responsible for designing the assays and control. The entire experiment can be performed in 3 h with single-beam spectrophotometers that are currently used in most undergraduate teaching laboratories. This exercise is suitable for large undergraduate classes of over 200 students and can be performed either by a single student or a student pair.

Immediate effects of anticancer drugs on mitochondrial oxygen consumption

The evolving role of mitochondria as a target for many anticancer drugs (e.g. platinum-based compounds, alkylating agents and anthracyclines) prompted us to investigate their immediate effects on the mitochondrial respiratory chain. For this purpose, we used a phosphorescence analyzer that measures [O 2] in solution. The [O 2] of solutions containing an appropriate substrate and various cell lines, tumors from patients or beef heart submitochondrial particles (SMPs) declined almost linearly ( r>0.99) as a function of time, indicating that the kinetics of cellular oxygen consumption were zero order. Rotenone inhibited respiration, confirming that oxygen was consumed by the respiratory chain. Exposure to a clinically relevant concentration of cisplatin (5 μM at 37° for 1–3 hr) had no effect on the respiration in cells or in SMP. Higher cisplatin concentrations (10–99 μM at 37° for 1–3 hr) produced <25% inhibition. Incubations with 4-hydroperoxycyclophosphamide (50–100 μM at 37° for 1 hr) inhibited oxygen consumption in SMP (∼70% inhibition at 50 μM) and in cells (∼30% inhibition at 50 μM). Incubations (37° for 1 hr) of SMP with doxorubicin (25–100 μM) and daunorubicin (25–100 μM) had no inhibitory effect on the respiration. By contrast, incubations (37° for 1 hr) of cells with doxorubicin (5–20 μM) and daunorubicin (2–20 μM) produced significant inhibition. We conclude that cisplatin does not directly damage the energy converting mechanism of mitochondria. On the other hand, comparable exposures to alkylating agents and anthracyclines produce immediate and dose-dependent impairment of cellular respiration.

Reactive oxygen species generated from the mitochondrial electron transport chain induce cytochrome c dissociation from beef-heart submitochondrial particles via cardiolipin peroxidation. Possible role in the apoptosis.

Cytochrome c release from mitochondria is a critical event in the apoptosis induction. Dissociation of cytochrome c from the mitochondrial inner membrane (IMM) is a necessary first step for cytochrome c release. In the present study, the effect of reactive oxygen species (ROS) on the dissociation of cytochrome c from beef-heart submitochondrial particles (SMP) and on the cardiolipin content was investigated. Exposure of SMP to mitochondrial-mediated ROS generation resulted in a large dissociation of cytochrome c from SMP and in a parallel loss of cardiolipin. Both these effects were directly and significantly correlated and also abolished by superoxide dismutase+catalase. These results demonstrate that ROS generation induces the dissociation of cytochrome c from IMM via cardiolipin peroxidation. The data may prove useful in clarifying the molecular mechanism underlying the release of cytochrome c from the mitochondria to the cytosol.

Inhibition of steady-state mitochondrial ATP synthesis by bicarbonate, an activating anion of ATP hydrolysis

Bicarbonate, an activating anion of ATP hydrolysis, inhibited ATP synthesis coupled to succinate oxidation in beef heart submitochondrial particles but diminished the lag time and increased the steady-state velocity of the 32Pi–ATP exchange reaction. The latter effects exclude the possibility that bicarbonate is inducing an intrinsic uncoupling between ATP hydrolysis and proton translocation at the level of F 1F o ATPase. The inhibition of ATP synthesis was competitive with respect to ADP at low fixed [Pi], mixed at high [Pi] and non-competitive towards Pi at any fixed [ADP]. From these results we can conclude that (i) bicarbonate does not bind to a Pi site in the mitochondrial F 1; (ii) it competes with the binding of ADP to a low-affinity site, likely the low-affinity non-catalytic nucleotide binding site. It is postulated that bicarbonate stimulates ATP hydrolysis and inhibits ATP synthesis by modulating the relative affinities of the catalytic site for ATP and ADP.

Reactive oxygen species generated by the mitochondrial respiratory chain affect the complex III activity via cardiolipin peroxidation in beef-heart submitochondrial particles

The aim of this study was to investigate the effect of reactive oxygen species (ROS), produced by the mitochondrial respiratory chain, on the activity of complex III and on the cardiolipin content in bovine-heart submitochondrial particles (SMP). ROS were produced by treatment of nicotinamide adenine dinucleotide (NADH) respiring SMP with rotenone. This treatment resulted in a production of superoxide anion, detected by the epinephrine method, which was blocked by superoxide dismutase (SOD). Exposure of SMP to mitochondrial-mediated ROS generation resulted in a marked loss of complex III activity and in a parallel loss of mitochondrial cardiolipin content. Both these effects were completely abolished by SOD+catalase. Exogenous added cardiolipin was able to almost completely prevent the ROS-mediated loss of complex III activity. No effect was obtained with other major phospholipid components of the mitochondrial membrane such as phosphatidylcholine and phosphatidylethanolamine, or with peroxidized cardiolipin. The results demonstrate that mitochondrial-mediated ROS generation affects the activity of complex III via peroxidation of cardiolipin, which is required for the functioning of this multisubunit enzyme complex. These results may prove useful in probing molecular mechanisms of ROS-induced peroxidative damage to mitochondria, which have been proposed to contribute to those physiopathological conditions characterized by an increase in the basal production of ROS such as aging, ischemia/reperfusion and chronic degenerative diseases.

Molecular characterisation of a NADH ubiquinone oxidoreductase subunit 5 from Schistosoma mansoni and inhibition of mitochondrial respiratory chain function by testosterone.

Complementary DNA, encoding the mitochondrial enzyme NADH-ubiquinone oxidoreductase subunit 5 (SmND5) of the human parasite Schistosoma mansoni was isolated by screening a S. mansoni cDNA library with a human androgen receptor (hAR) cDNA probe. The complete nucleotide and deduced aminoacid sequences of SmND5 were determined. Southern blot analysis revealed the occurrence of a single copy gene for SmND5 and by means of RT-PCR, it was shown that sex- and stage-specific expression of SmND5 occurred. In order to establish a functional relationship between the mitochondrial enzyme and the androgen receptor, the effects of testosterone were compared to those of classical respiratory chain inhibitors, using adult schistosome and beef heart submitochondrial particles. Physiological concentrations of testosterone were able to inhibit the maintenance of proton gradient across the mitochondrial membranes, as well as ATP synthesis. The steroid was found to be cytotoxic to the larvae, but not to adult schistosomes. A model is proposed to explain the observed in vivo testosterone-related differences in worm burdens, in experimental chronic infections.

On the Mechanism of Quinol Oxidation in thebc1 Complex

The question of whether significant levels of a semiquinone can be generated in the Qo site of the bc1 complex under conditions of oxidant-induced reduction is relevant to the mechanism of bifurcation of electron transfer in this site. It has already been reported that beef heart submitochondrial particles under such conditions exhibit an EPR-detectable semiquinone, which is distinct from Q-i and which was attributed to a semiquinone in the Qo site (de Vries, S., Albracht, S. P. J., Berden, J. A., and Slater, E. C. (1981) J. Biol. Chem. 256, 11996-11998). However, we show here that this signal, which can be generated to a level of around 0.1 per bc1 monomer, is insensitive to the Qo site inhibitors myxothiazol, E-beta-methoxyacrylate-stilbene, and stigmatellin, indicating that it does not arise from a Q-o species. Based on sensitivities to inhibitors of other Q sites, up to 60% of the signal may arise from semiquinones of complexes I and II. We further show that the iron-sulfur center remains EPR silent under oxidant-induced reduction conditions. Overall, the results indicate that, under conditions of oxidant-induced reduction, the Qo site is occupied primarily by quinol with the iron-sulfur center oxidized, or, possibly, by an antiferromagnetically coupled semiquinone/reduced iron-sulfur center pair, which are EPR silent. This is discussed in relation to proposed mechanisms of quinol oxidation in the Qo site, and we describe a minimal intermediate-controlled bifurcation model based on rate constants by which bifurcated electron transfer at the Qo site might occur.

Relevance of divalent cations to ATP-driven proton pumping in beef heart mitochondrial F0F1-ATPase.

The ATP hydrolysis rate and the ATP hydrolysis-linked proton translocation by the F0F1-ATPase of beef heart submitochondrial particles were examined in the presence of several divalent metal cations. All Me-ATP complexes tested sustained ATP hydrolysis, although to a different extent. However, only Mg- and Mn-ATP-dependent hydrolysis could sustain a high level of proton pumping activity, as determined by acridine fluorescence quenching. Moreover, the Km of the Me-ATP hydrolysis-induced proton pumping activity was very similar to the Km value of Me-ATP hydrolysis. Both oligomycin and DCCD caused the full recovery of the fluorescence, providing clear evidence for the association of Mg-ATP hydrolysis with proton translocation through the F0F1-ATPase complex. In contrast, with other Me-ATP complexes, including Ca-ATP as substrate, the proton pumping activity was undetectable, implicating an uncoupling nature for these substrates. Attempts to demonstrate the involvement of the epsilon subunit of the enzyme in the coupling mechanism failed, suggesting that the participation of at least the N-terminal segment of the subunit in the coupling mechanism of the mitochondrial enzyme is unlikely.

The effect of the neuroblastoma-seeking agent meta-iodobenzylguanidine (MIBG) on NADH-driven superoxide formation and NADH-driven lipid peroxidation in beef heart submitochondrial particles

In this paper we report the effects of the neuroblastoma-seeking agent meta-iodobenzylguanidine (MIBG) on NADH-driven superoxide formation and NADH-driven lipid peroxidation in beef heart submitochondrial particles. MIBG is a structural analogue of noradrenaline and is capable of inhibiting complex I and complex III of the respiratory chain. The results of our studies show that MIBG enhanced both NADH-driven superoxide formation and NADH-driven lipid peroxidation at concentrations that are likely to exist inside mitochondria of the target cells of neuroblastoma patients treated with [ 131I]MIBG. The effect of MIBG is comparable to that of rotenone (an inhibitor of complex I) rather than that of antimycin (an inhibitor of complex III). These results suggest that the formation of superoxide and lipid peroxidation contributes to the cytotoxicity of [ 131I]MIBG.

The Effect of the Neuroblastoma-seeking Agent meta-Iodobenzylguanidine (MIBG) on NADH-driven Superoxide Formation and NADH-driven Lipid Peroxidation in Beef Heart Submitochondrial Particles

The Effect of the Neuroblastoma-seeking Agent meta-Iodobenzylguanidine (MIBG) on NADH-driven Superoxide Formation and NADH-driven Lipid Peroxidation in Beef Heart Submitochondrial Particles

Pro- and anti-oxidant activities of the mitochondrial respiratory chain: factors influencing NAD(P)H-induced lipid peroxidation

This paper is a study of factors influencing the rate of lipid peroxidation in beef heart submitochondrial particles induced by NAD(P)H via the NADH-ubiquinone oxidoreductase (Complex I) of the respiratory chain. In accordance with earlier observations, both NADH and NADPH initiated lipid peroxidation in the presence of ADP-Fe 3+. The rate of the reaction, measured as oxygen consumption and formation of thiobarbituric acid reactive substances, was biphasic as a function of NADH concentration, reaching a maximum at low NADH concentrations and then declining. In contrast, the NADPH-initiated lipid peroxidation showed a monophasic concentration profile of hyperbolic character. Rotenone did not eliminate the biphasicity of the NADH-induced reaction, indicating that this was not due to an antioxidant effect of reduced ubiquinone at high NADH concentrations. This conclusion was further supported by the demonstration that extraction of ubiquinone from the particles did not relieve the inhibition of lipid peroxidation by high NADH concentrations. However rhein, another inhibitor of Complex I, eliminated the biphasicity, and even caused a substantial stimulation of the NADH-induced lipid peroxidation in the particles upon extraction of ubiquinone by pentane. No similar effect occurred in the case of NADPH-induced lipid peroxidation. Furthermore, rhein facilitated both NADH- and NADPH-induced lipid peroxidation even in the absence of added ADP-Fe 3+, in a fashion similar to that earlier reported with succinate in the presence of theonyltrifluoroacetone. Based on these findings and measurements of the redox states of ubiquinone and cytochromes in the presence of KCN and NADH or NADPH, it is concluded that Complex I may distinguish between electron input from NADH and NADPH by differences in the site(s) of substrate binding and in the pathways and rates of NADH and NADPH oxidation.

Lipid peroxidation and changes in the ubiquinone content and the respiratory chain enzymes of submitochondrial particles

The relationship between lipid peroxidation induced by ascorbate and adenosine ADP/Fe 3+, and its effect on the respiratory chain activities of beef heart submitochondrial particles has been investigated. Lipid peroxidation, measured as thiobarbituric acid reactive substance formation, resulted in an inhibition of the NADH and succinate oxidase activities. Examination of several partial reactions of the respiratory chain revealed inactivation primarily of those involving endogenous ubiquinone, i.e., NADH- and succinate-ubiquinone 1 and cytochrome c reductases. Ubiquinol–cytochrome c reductase, measured with reduced ubiquinone 2 as electron donor, was unaffected. The amount of NADH- or succinate-reducible cytochrome b in the presence of cyanide was strongly decreased, but could be recovered by the addition of antimycin. There occurred a substantial decrease of the ubiquinone content in the course of lipid peroxidation, with a linear relationship between this decrease and the NADH and succinate oxidase activities. The results are consistent with the conclusion that the ubiquinone pool undergoes an oxidative modification during lipid peroxidation, to a form that can no longer function as a component of the respiratory chain. Lipid peroxidation also led to a partial inhibition of the succinate dehydrogenase and cytochrome c oxidase activities and a minor decrease of the cytochrome c and cytochrome a contents. Reduction of endogenous ubiquinone prevented lipid peroxidation as well as the concomitant modification of ubiquinone and inactivation of the respiratory chain. These observations suggest that the destruction of ubiquinone through lipid peroxidation is the primary cause of inactivation of the respiratory chain, and emphasize the antioxidant role of ubiquinol in preventing these effects. The possible implications of these findings for regulation of the cellular turnover of ubiquinone by the prevailing oxidative stress are discussed. Copyright © 1996 Elsevier Science Inc.

Oxygenation of biomembranes by mammalian lipoxygenases: The role of ubiquinone

15-Lipoxygenase is implicated in the selective breakdown of mitochondria during red cell maturation by virtue of its capability of directly oxygenating phospholipids. To address the reason of the selectivity for mitochondria, we studied the reaction of pure rabbit 15-lipoxygenase with beef heart submitochondrial particles in vitro. This reaction is characterised by a loss of polyenoic fatty acids, the formation of phospholipid-bound hydroperoxy- and keto-polyenoic fatty acids, and oxidative modification of membrane proteins. The total oxygen uptake exceeds the formation of oxygenated polyenoic fatty acids several times. The excessive oxygen uptake was not inhibited by 3,5-di-tert-butyl-4-hydroxytoluene or by respiratory inhibitors, but was partly suppressed by superoxide dismutase plus catalase, salicylate, or mannitol. Pentane-extraction of the submitochondrial particles abolished the excessive oxygen uptake, whereas reconstitution with ubiquinone-50 restored it. A marked excessive oxygen uptake did not occur during the analogous reaction with erythrocyte ghosts. It is proposed that ubiquinone-50 triggers the formation of hydroxyl radicals from 15-lipoxygenase-derived hydroperoxy-lipids via a Fenton-type reaction driven by ubisemiquinone radicals. A new prooxidative function of ubiquinone in the biologically programmed degradation of mitochondria in certain types of cells is proposed.

Endogenous ubiquinol prevents protein modification accompanying lipid peroxidation in beef heart submitochondrial particles

This article is a study of the relationship between lipid peroxidation and protein modification in beef heart submitochondrial particles, and the protective effect of endogenous ubiquinol (reduced coenzyme Q) against these effects. ADP-Fe 3· and ascorbate were used to initiate lipid peroxidation and protein modification, which were monitored by measuring TBARS and protein carbonylation, respectively. Endogenous ubiquinone was reduced by the addition of succinate and antimycin. The parameters investigated included extraction and reincorporation of ubiquinone, and comparison of the effect of ubiquinol with those of various antioxidant compounds and enzymes, as well as the iron chelator EDTA. Under all conditions employed there was a close correlation between lipid peroxidation and protein carbonylation, and the inhibition of these effects by endogenous ubiquinol. SDS-PAGE analysis revealed a differential effect on individual protein components and its prevention by ubiquinol. Conceivable mechanisms behind the observed oxidative modifications of membrane phospholipids and proteins and of the role of ubiquinol in preventing these effects are considered.

DBHBM (3,5-Dibromo-4-Hydroxy-Benzylidenemalonitrile) Is a Novel Inhibitor of Electron-Transfer Through the QN Center of the Mitochondrial bc1 Complex

DBHBM (3,5-dibromo-4-hydroxy-benzylidenemalonitrile) inhibited the NADH- or succinate-supported rate of O2 consumption in beef heart submitochondrial particles (Ki = 7 × 10−7 M). Oxygen comsumption was restored with the addition of ascorbate/TMPD, indicating that the inhibitory effect was on the ubiquinol-cytochrome c reductase activity of the respiratory chain. Difference spectra with submitochondrial particles indicated that DBHBM blocked electron transport through the cytochrome bc1 complex, in a mode closely similar to that of antimycin A. The reduction rates of cytochrome b by succinate were strongly inhibited in the presence of DBHBM plus myxothiazol, but not by DBHBM plus antimycin A. These data suggest that DBHBM may bind primarily to the QN center. In the purified bc1 complex, DBHBM and antimycin A induced a red shift from 562 to 566 nm of the α peak of cytochrome b, supporting the idea that DBHBM influences predominantly the ligand field of the b562 (bh) heme. Difference spectra in the presence or absence of myxothiazol showed that DBHBM induced the same red shift with a maximum at 565 nm and a minimum at 559 nm. We conclude that DBHBM blocks electron transfer at the QN center and thus may be considered a novel group III inhibitor of the bc1 complex.

Hydroxyl Radical Generation During Mitochondrial Electron-Transfer and the Formation of 8-Hydroxydesoxyguanosine in Mitochondrial-DNA

Production of hydroxyl radicals (HO.) by substrate-supplemented beef heart submitochondrial particles was studied by electron paramagnetic resonance in conjunction with the spin trap 5,5′-dimethyl-1-pirroline-N-oxide (DMPO). Supplementation of submitochondrial particles with NADH or succinate in the presence of antimycin resulted in the formation hydroxyl-, α-hydroxyethyl-, and methyl radical adducts. The latter two adducts were derived from HO. attack of ethanol or dimethyl sulfoxide (DMSO), respectively, the solvents used for the inhibitors of the respiratory chain. These ESR signals were slightly increased by superoxide dismutase and abolished by catalase. Further support for the production of HO. during mitochondrial electron transfer was furnished by kinetic competition experiments with DMSO as the HO. scavenger. This approach yielded a kSCAVENGER/kDMPO value of 1.7, in agreement with a competitive spin trapping of free HO. using DMSO as a scavenger. The scission of H2O2 to HO. requires consideration of a Fenton chemistry, i,e., the participation of metals or redox active metal pools in mitochondria to drive this reaction, The effect of several metal chelators on the formation of both HO. and H2O2 was examined. Bathophenantroline, bathocuproine, and desferrioxamine decreased the DMPO-HO. signal and increased accumulation of H2O2. Conversely, EDTA or diethylenetriaminepentaacetic acid substantially increased the DMPO-HO. signal intensity and decreased H2O2 accumulation. These different results were rationalized in terms of the reduction potential of the redox couples involved, i,e., that of the ligated metal and those encompassed in the one-electron reduction of superoxide radical and of hydrogen peroxide. The formation of 8-hydroxydesoxyguanosine in mitochondrial DNA was examined under experimental conditions in which H2O2 production by isolated mitochondria was enhanced. The formation of 8-hydroxydesoxyguanosine increased with increasing rates of H2O2 formation. The biological significance of H2O2 and HO. formation during mitochondrial electron transfer is discussed in terms of oxidative damage of mitochondrial DNA and the implications for mitochondrial functions and aging.