Respiratory chain components involved in the glycerophosphate dehydrogenase-dependent ROS production by brown adipose tissue mitochondria

Involvement of mammalian mitochondrial glycerophosphate dehydrogenase (mGPDH, EC 1.1.99.5) in reactive oxygen species (ROS) generation was studied in brown adipose tissue mitochondria by different spectroscopic techniques. Spectrofluorometry using ROS-sensitive probes CM-H2DCFDA and Amplex Red was used to determine the glycerophosphate- or succinate-dependent ROS production in mitochondria supplemented with respiratory chain inhibitors antimycin A and myxothiazol. In case of glycerophosphate oxidation, most of the ROS originated directly from mGPDH and coenzyme Q while complex III was a typical site of ROS production in succinate oxidation. Glycerophosphate-dependent ROS production monitored by KCN-insensitive oxygen consumption was highly activated by one-electron acceptor ferricyanide, whereas succinate-dependent ROS production was unaffected. In addition, superoxide anion radical was detected as a mGPDH-related primary ROS species by fluorescent probe dihydroethidium, as well as by electron paramagnetic resonance (EPR) spectroscopy with DMPO spin trap. Altogether, the data obtained demonstrate pronounced differences in the mechanism of ROS production originating from oxidation of glycerophosphate and succinate indicating that electron transfer from mGPDH to coenzyme Q is highly prone to electron leak and superoxide generation.     

ROS generation and multiple forms of mammalian mitochondrial glycerol-3-phosphate dehydrogenase

Overproduction of reactive oxygen species (ROS) has been implicated in a range of pathologies. Mitochondrial flavin dehydrogenases glycerol-3-phosphate dehydrogenase (mGPDH) and succinate dehydrogenase (SDH) represent important ROS source, but the mechanism of electron leak is still poorly understood. To investigate the ROS production by the isolated dehydrogenases, we used brown adipose tissue mitochondria solubilized by digitonin as a model. Enzyme activity measurements and hydrogen peroxide production studies by Amplex Red fluorescence, and luminol luminescence in combination with oxygraphy revealed flavin as the most likely source of electron leak in SDH under in vivo conditions, while we propose coenzyme Q as the site of ROS production in the case of mGPDH. Distinct mechanism of ROS production by the two dehydrogenases is also apparent from induction of ROS generation by ferricyanide which is unique for mGPDH. Furthermore, using native electrophoretic systems, we demonstrated that mGPDH associates into homooligomers as well as high molecular weight supercomplexes, which represent native forms of mGPDH in the membrane. By this approach, we also directly demonstrated that isolated mGPDH itself as well as its supramolecular assemblies are all capable of ROS production.     

Glycerophosphate-dependent hydrogen peroxide production by brown adipose tissue mitochondria and its activation by ferricyanide

Oxidation of glycerophosphate (GP) by brown adipose tissue mitochondria in the presence of antimycin A was found to be accompanied by significant production of hydrogen peroxide. GP-dependent hydrogen peroxide production could be detected by p-hydroxyphenylacetate fluorescence changes or as an antimycin A-insensitive oxygen consumption. One-electron acceptor, potassium ferricyanide, highly stimulated the rate of GP-dependent antimycin A-insensitive oxygen uptake, which was prevented by inhibitors of mitochondrial GP dehydrogenase (mGPDH) or by coenzyme Q(CoQ). GP-dependent ferricyanide-induced peroxide production was also determined luminometrically, using mitochondria or partially purified mGPDH. Ferricyanide-induced peroxide production was negligible, when succinate or NADH was used as a substrate. These results indicate that hydrogen peroxide is produced directly by mGPDH and reflect the differences in the transport of reducing equivalents from mGPDH and succinate dehydrogenase to the CoQ pool. The data suggest that more intensive production of reactive oxygen species may be present in mammalian cells with active mGPDH.     

Inhibition of glycerophosphate-dependent H2O2 generation in brown fat mitochondria by idebenone

The established protective effect of coenzyme Q (CoQ) analogs is dependent on the location of reactive oxygen species (ROS) generation. One of these analogs--idebenone (hydroxydecyl-ubiquinone) is used as an antioxidative therapeutic drug. We tested its scavenging effect on the glycerophosphate (GP)-dependent ROS production as this enzyme was shown as a new site in the mitochondrial respiratory chain where ROS can be generated. We observed that idebenone inhibits both GP- and succinate-dependent ROS production. Idebenone and CoQ1 were found to be more efficient in the scavenging activity (IC50: 0.052 and 0.075 microM, respectively) than CoQ3 (IC50: 45.8 microM). Idebenone also inhibited ferricyanide (FeCN)-activated, GP-dependent ROS production. Our data thus extend previous findings on the scavenging effect of idebenone and show that it can also eliminate GP-dependent ROS generation.     

High efficiency of ROS production by glycerophosphate dehydrogenase in mammalian mitochondria

We investigated hydrogen peroxide production in mitochondria with low (liver, heart, brain) and high (brown adipose tissue, BAT) content of glycerophosphate dehydrogenase (mGPDH). ROS production at state 4 due to electron backflow from mGPDH was low, but after inhibition of electron transport with antimycin A high rates of mGPDH-dependent ROS production were observed in liver, heart and brain mitochondria. When this ROS production was related to activity of mGPDH, many-fold higher ROS production was found in contrast to succinate- (39-, 28-, 3-fold) or pyruvate plus malate-dependent ROS production (32-, 96-, 5-fold). This specific rate of mGPDH-dependent ROS production was also exceedingly higher (28-, 66-, 22-fold) compared to that in BAT. mGPDH-dependent ROS production was localized to the dehydrogenase + CoQ and complex III, the latter being the highest in all mitochondria but BAT. Our results demonstrate high efficiency of mGPDH-dependent ROS production in mammalian mitochondria with a low content of mGPDH and suggest its endogenous inhibition in BAT.     

The intracellular tyrosine residues of the ATP-gated P2X(1) ion channel are essential for its function

In order to gain a first insight into the effects of reactive oxygen species (ROS) on plant mitochondria, we studied the effect of the ROS producing system consisting of xanthine plus xanthine oxidase on the rate of membrane potential (DeltaPsi) generation due to either succinate or NADH addition to durum wheat mitochondria as monitored by safranin fluorescence. We show that the early ROS production inhibits the succinate-dependent, but not the NADH-dependent, DeltaPsi generation and oxygen uptake. This inhibition appears to depend on the impairment of mitochondrial permeability to succinate. It does not involve mitochondrial thiol groups sensitive to either mersalyl or N-ethylmaleimide and might involve both protein residues and/or membrane lipids, as suggested by the mixed nature. We propose that, during oxidative stress, early generation of ROS can affect plant mitochondria by impairing metabolite transport, thus preventing further substrate oxidation, DeltaPsi generation and consequent large-scale ROS production.     

Cadmium inhibits the electron transfer chain and induces reactive oxygen species

Recent research indicates that cadmium (Cd) induces oxidative damage in cells; however, the mechanism of the oxidative stress induced by this metal is unclear. We investigated the effects of Cd on the individual complexes of the electron transfer chain (ETC) and on the stimulation of reactive oxygen species (ROS) production in mitochondria. The activity of complexes II (succinate:ubiquinone oxidoreductase) and III (ubiquinol:cytochrome c oxidoreductase) of mitochondrial ETC from liver, brain, and heart showed greater inhibition by Cd than the other complexes. Cd stimulated ROS production in the mitochondria of all three tissues mentioned above. The effect of various electron donors (NADH, succinate, and 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinol) on ROS production was tested separately in the presence and in the absence of Cd. ESR showed that complex III might be the only site of ROS production induced by Cd. The results of kinetic studies and electron turnover experiments suggest that Cd may bind between semiubiquinone and cytochrome b566 of the Q0 site of cytochrome b of complex III, resulting in accumulation of semiubiquinones at the Q0 site. The semiubiquinones, being unstable, are prone to transfer one electron to molecular oxygen to form superoxide, providing a possible mechanism for Cd-induced generation of ROS in mitochondria.     

A Refined Analysis of Superoxide Production by Mitochondrial sn-Glycerol 3-Phosphate Dehydrogenase

The oxidation of sn-glycerol 3-phosphate by mitochondrial sn-glycerol 3-phosphate dehydrogenase (mGPDH) is a major pathway for transfer of cytosolic reducing equivalents to the mitochondrial electron transport chain. It is known to generate H₂O₂ at a range of rates and from multiple sites within the chain. The rates and sites depend upon tissue source, concentrations of glycerol 3-phosphate and calcium, and the presence of different electron transport chain inhibitors. We report a detailed examination of H₂O₂ production during glycerol 3-phosphate oxidation by skeletal muscle, brown fat, brain, and heart mitochondria with an emphasis on conditions under which mGPDH itself is the source of superoxide and H₂O₂. Importantly, we demonstrate that a substantial portion of H₂O₂ production commonly attributed to mGPDH originates instead from electron flow through the ubiquinone pool into complex II. When complex II is inhibited and mGPDH is the sole superoxide producer, the rate of superoxide production depends on the concentrations of glycerol 3-phosphate and calcium and correlates positively with the predicted reduction state of the ubiquinone pool. mGPDH-specific superoxide production plateaus at a rate comparable with the other major sites of superoxide production in mitochondria, the superoxide-producing center shows no sign of being overreducible, and the maximum superoxide production rate correlates with mGPDH activity in four different tissues. mGPDH produces superoxide approximately equally toward each side of the mitochondrial inner membrane, suggesting that the Q-binding pocket of mGPDH is the major site of superoxide generation. These results clarify the maximum rate and mechanism of superoxide production by mGPDH.     

Effect of KCl-medium on succinate oxidation and hydrogen peroxide generation in mitochondria of sugar beet taproot

The effect of substitution of KCl for sucrose in the reaction medium on succinate oxidation and hydrogen peroxide generation was investigated in the mitochondria isolated from stored taproots of sugar beet (Beta vulgaris L.). In a sucrose-containing medium, oxidation of succinate was inhibited by oxaloacetate; this inhibition was especially pronounced upon a decrease in substrate concentration and eliminated in the presence of glutamate, which removed oxaloacetate in the course of transamination. Irrespective of succinate concentration, substitution of KCl for sucrose in the medium considerably enhanced suppression of succinate oxidation apparently as a result of slow activation of succinate dehydrogenase (SDH) by its substrate. In this case, mitochondria showed the symptoms of uncoupling, lower values of membrane potential (ΔΨ), respiratory control (RC), and ADP/O induced by electrophoretic transport of potassium via K⁺ channel of mitochondria. KCl-dependent suppression of succinate oxidation by taproot mitochondria was accompanied by a considerable inhibition of H₂O₂ production as compared with the sucrose-containing medium. These results indicate that in the presence of potassium ions, ΔΨ dissipates, suppression of succinate oxidation by oxaloacetate increases, and succinate-dependent generation of ROS in sugar beet mitochondria is inhibited. A possible physiological role of oxaloacetate-restricted SDH activity in the suppression of respiration of storage organs protecting mitochondria from oxidative stress is discussed.     

Mitochondrial fatty acid oxidation and oxidative stress: Lack of reverse electron transfer-associated production of reactive oxygen species

Reverse electron transfer (RET) from succinate to NAD⁺ is known to be accompanied by high generation of reactive oxygen species (ROS). In contrast, oxidation of fatty acids by mitochondria, despite being a powerful source of FADH₂, does not lead to RET-associated high ROS generation. Here we show that oxidation of carnitine esters of medium- and long-chain fatty acids by rat heart mitochondria is accompanied by neither high level of NADH/NAD⁺ nor intramitochondrial reduction of acetoacetate to β-hydroxybutyrate, comparable to those accompanying succinate oxidation, although it produces the same or higher energization of mitochondria as evidenced by high transmembrane potential. Also in contrast to the oxidation of succinate, where conversion of the pH difference between the mitochondrial matrix and the medium into the transmembrane electric potential by addition of nigericin results in a decrease of ROS generation, the same treatment during oxidation of octanoylcarnitine produces a large increase of ROS. Analysis of respiratory chain complexes by Blue Native polyacrylamide gel electrophoresis revealed bands that could tentatively point to supercomplex formation between complexes II and I and complexes II and III. However, no such association could be found between complex I and the electron transferring flavoprotein that participates in fatty acid oxidation. It is speculated that structural association between respective respiratory chain components may facilitate effective reverse electron transfer.     

Fatty acids decrease mitochondrial generation of reactive oxygen species at the reverse electron transport but increase it at the forward transport

Long-chain nonesterified (“free”) fatty acids (FFA) can affect the mitochondrial generation of reactive oxygen species (ROS) in two ways: (i) by depolarisation of the inner membrane due to the uncoupling effect and (ii) by partly blocking the respiratory chain. In the present work this dual effect was investigated in rat heart and liver mitochondria under conditions of forward and reverse electron transport. Under conditions of the forward electron transport, i.e. with pyruvate plus malate and with succinate (plus rotenone) as respiratory substrates, polyunsaturated fatty acid, arachidonic, and branched-chain saturated fatty acid, phytanic, increased ROS production in parallel with a partial inhibition of the electron transport in the respiratory chain, most likely at the level of complexes I and III. A linear correlation between stimulation of ROS production and inhibition of complex III was found for rat heart mitochondria. This effect on ROS production was further increased in glutathione-depleted mitochondria. Under conditions of the reverse electron transport, i.e. with succinate (without rotenone), unsaturated fatty acids, arachidonic and oleic, straight-chain saturated palmitic acid and branched-chain saturated phytanic acid strongly inhibited ROS production. This inhibition was partly abolished by the blocker of ATP/ADP transfer, carboxyatractyloside, thus indicating that this effect was related to uncoupling (protonophoric) action of fatty acids. It is concluded that in isolated rat heart and liver mitochondria functioning in the forward electron transport mode, unsaturated fatty acids and phytanic acid increase ROS generation by partly inhibiting the electron transport and, most likely, by changing membrane fluidity. Only under conditions of reverse electron transport, fatty acids decrease ROS generation due to their uncoupling action.     

Reactive oxygen and targeted antioxidant administration in endothelial cell mitochondria

We used fluorescent probes and EPR to study the mechanism(s) underlying reactive oxygen species (ROS) production by endothelial cell mitochondria and the action of mitoquinol, a mitochondria-targeted antioxidant. ROS measured by fluorescence resulted from complex I superoxide released to the matrix and converted to H(2)O(2). In contrast, EPR largely detected superoxide generated at complex III and effluxed outward. ROS fluorescence by mitochondria fueled by the complex II substrate, succinate, was substantial but markedly inhibited by rotenone. Superoxide, detected by EPR, in succinate-fueled mitochondria was not inhibited by rotenone and likely derived from semiquinone formation at complex III. Mitoquinol decreased H(2)O(2) fluorescence by succinate-fueled mitochondria but had little effect on the EPR signal for superoxide. This was not associated with a detectable decrease in membrane potential. Mitoquinol markedly enhanced ROS fluorescence in mitochondria fueled by the complex I substrates, glutamate and malate. Inhibitor studies suggested that this occurred in complex I, at one or more Q binding pockets. The above effects of mitoquinol were determined in mitochondria isolated and subsequently exposed to the targeted antioxidant. However, similar effects were observed in mitochondria after antecedent exposure to mitoquinol/mitoquinone in culture, suggesting that the agent is retained after isolation of the organelles. In conclusion, ROS production in bovine aortic endothelial cell mitochondria results largely from reverse transport to complex I and through the Q cycle in complex III. Mitoquinol blocks ROS from reverse electron transport but increases superoxide production derived from forward transport. These effects likely occur at one or more Q binding sites in complex I.     

Glycerophosphate-dependent hydrogen peroxide production by rat liver mitochondria

We studied the extent to which hormonally-induced mitochondrial glycerophosphate dehydrogenase (mGPDH) activity contributes to the supply of reducing equivalents to the mitochondrial respiratory chain in the rat liver. The activity of glycerophosphate oxidase was compared with those of NADH oxidase and/or succinate oxidase. It was found that triiodothyronine-activated mGPDH represents almost the same capacity for the saturation of the respiratory chain as Complex II. Furthermore, the increase of mGPDH activity induced by triiodothyronine correlated with an increase of capacity for glycerophosphate-dependent hydrogen peroxide production. As a result of hormonal treatment, a 3-fold increase in glycerophosphate-dependent hydrogen peroxide production by liver mitochondria was detected by polarographic and luminometric measurements.     

Mitochondrial superoxide and coenzyme Q in insulin-deficient rats: increased electron leak

Mitochondrial superoxide is important in the pathogeneses of diabetes and its complications. However, there is uncertainty regarding the intrinsic propensity of mitochondria to generate this radical. Studies to date suggest that superoxide production by mitochondria of insulin-sensitive target tissues of insulin-deficient rodents is reduced or unchanged. Moreover, little is known of the role of the Coenzyme Q (CoQ), whose semiquinone form reacts with molecular oxygen to generate superoxide. We measured reactive oxygen species (ROS) production, respiratory parameters, and CoQ content in mitochondria from gastrocnemius muscle of control and streptozotocin (STZ)-diabetic rats. CoQ content did not differ between mitochondria isolated from vehicle- or STZ-treated animals. CoQ also was unaffected by weight loss in the absence of diabetes (induced by caloric restriction). Under state 4 or state 3 conditions, both respiration and ROS release were reduced in diabetic mitochondria fueled with succinate, glutamate plus malate, or with all three substrates (continuous TCA cycle). However, H(2)O(2) and directly measured superoxide production were substantially increased in gastrocnemius mitochondria of diabetic rats when expressed per unit oxygen consumed. On the basis of substrate and inhibitor effects, the mechanism involved multiple electron transport sites. More limited results using heart mitochondria were similar. ROS per unit respiration was greater in muscle mitochondria from diabetic compared with control rats during state 3, as well as state 4, while the reduction in ROS per unit respiration on transition to state 3 was less for diabetic mitochondria. In summary, ROS production is, in fact, increased in mitochondria from insulin-deficient muscle when considered relative to electron transport. This is evident on multiple energy substrates and in different respiratory states. CoQ is not reduced in diabetic mitochondria or with weight loss due to food restriction. The implications of these findings are discussed.     

Generation of superoxide by the mitochondrial Complex I

Superoxide production by inside-out coupled bovine heart submitochondrial particles, respiring with succinate or NADH, was measured. The succinate-supported production was inhibited by rotenone and uncouplers, showing that most part of superoxide produced during succinate oxidation is originated from univalent oxygen reduction by Complex I. The rate of the superoxide (O2*-)) production during respiration at a high concentration of NADH (1 mM) was significantly lower than that with succinate. Moreover, the succinate-supported O2*- production was significantly decreased in the presence of 1 mM NADH. The titration curves, i.e., initial rates of superoxide production versus NADH concentration, were bell-shaped with the maximal rate (at 50 microM NADH) approaching that seen with succinate. Both NAD+ and acetyl-NAD+ inhibited the succinate-supported reaction with apparent Ki's close to their Km's in the Complex I-catalyzed succinate-dependent energy-linked NAD+ reduction (reverse electron transfer) and NADH:acetyl-NAD+ transhydrogenase reaction, respectively. We conclude that: (i) under the artificial experimental conditions the major part of superoxide produced by the respiratory chain is formed by some redox component of Complex I (most likely FMN in its reduced or free radical form); (ii) two different binding sites for NADH (F-site) and NAD+ (R-site) in Complex I provide accessibility of the substrates-nucleotides to the enzyme red-ox component(s); F-site operates as an entry for NADH oxidation, whereas R-site operates in the reverse electron transfer and univalent oxygen reduction; (iii) it is unlikely that under the physiological conditions (high concentrations of NADH and NAD+) Complex I is responsible for the mitochondrial superoxide generation. We propose that the specific NAD(P)H:oxygen superoxide (hydrogen peroxide) producing oxidoreductase(s) poised in equilibrium with NAD(P)H/NAD(P)+ couple should exist in the mitochondrial matrix, if mitochondria are, indeed, participate in ROS-controlled processes under physiologically relevant conditions.     

Q-site inhibitor induced ROS production of mitochondrial complex II is attenuated by TCA cycle dicarboxylates

The impact of complex II (succinate:ubiquinone oxidoreductase) on the mitochondrial production of reactive oxygen species (ROS) has been underestimated for a long time. However, recent studies with intact mitochondria revealed that complex II can be a significant source of ROS. Using submitochondrial particles from bovine heart mitochondria as a system that allows the precise setting of substrate concentrations we could show that mammalian complex II produces ROS at subsaturating succinate concentrations in the presence of Q-site inhibitors like atpenin A5 or when a further downstream block of the respiratory chain occurred. Upon inhibition of the ubiquinone reductase activity, complex II produced about 75% hydrogen peroxide and 25% superoxide. ROS generation was attenuated by all dicarboxylates that are known to bind competitively to the substrate binding site of complex II, suggesting that the oxygen radicals are mainly generated by the unoccupied flavin site. Importantly, the ROS production induced by the Q-site inhibitor atpenin A5 was largely unaffected by the redox state of the Q pool and the activity of other respiratory chain complexes. Hence, complex II has to be considered as an independent source of mitochondrial ROS in physiology and pathophysiology.     

Increased expression of mitochondrial glycerophosphate dehydrogenase and antioxidant enzymes in prostate cancer cell lines/cancer

The involvement of mitochondrial glycerophosphate dehydrogenase (mGPDH) has previously been established in the production of ROS in prostate cancer cell lines (LNCaP, DU145, PC3 and CL1). The current study demonstrates that the mRNA level of mGPDH in prostate cancer cells is 3.3-8.9-fold higher compared to the normal prostate epithelial cell line, PNT1A. This is consistent with the enzymatic activity and protein level of mGPDH. However, cytochrome c oxidase (COX) activity is 2.9-3.2-fold down-regulated in androgen-independent prostate cancer cell lines. The level of antioxidant enzymes, catalase, MnSOD and CuZnSOD are up-regulated in prostate cancer cell lines. Furthermore, it was observed that the activity of mGPDH is significantly higher in liver tissues from all mice with cancer compared to liver tissues from control mice. These data suggest that the up-regulation of mGPDH, due to a highly glycolytic environment, contributes to the overall increase in ROS generation and may result in the progression of the cancer.     

The role of external and matrix pH in mitochondrial reactive oxygen species generation

Reactive oxygen species (ROS) generation in mitochondria as a side product of electron and proton transport through the inner membrane is important for normal cell operation as well as development of pathology. Matrix and cytosol alkalization stabilizes semiquinone radical, a potential superoxide producer, and we hypothesized that proton deficiency under the excess of electron donors enhances reactive oxygen species generation. We tested this hypothesis by measuring pH dependence of reactive oxygen species released by mitochondria. The experiments were performed in the media with pH varying from 6 to 8 in the presence of complex II substrate succinate or under more physiological conditions with complex I substrates glutamate and malate. Matrix pH was manipulated by inorganic phosphate, nigericine, and low concentrations of uncoupler or valinomycin. We found that high pH strongly increased the rate of free radical generation in all of the conditions studied, even when DeltapH=0 in the presence of nigericin. In the absence of inorganic phosphate, when the matrix was the most alkaline, pH shift in the medium above 7 induced permeability transition accompanied by the decrease of ROS production. ROS production increase induced by the alkalization of medium was observed with intact respiring mitochondria as well as in the presence of complex I inhibitor rotenone, which enhanced reactive oxygen species release. The phenomena revealed in this report are important for understanding mechanisms governing mitochondrial production of reactive oxygen species, in particular that related with uncoupling proteins.     

Increased expression of mitochondrial glycerophosphate dehydrogenase and antioxidant enzymes in prostate cancer cell lines/cancer

The involvement of mitochondrial glycerophosphate dehydrogenase (mGPDH) has previously been established in the production of ROS in prostate cancer cell lines (LNCaP, DU145, PC3 and CL1). The current study demonstrates that the mRNA level of mGPDH in prostate cancer cells is 3.3–8.9-fold higher compared to the normal prostate epithelial cell line, PNT1A. This is consistent with the enzymatic activity and protein level of mGPDH. However, cytochrome c oxidase (COX) activity is 2.9–3.2-fold down-regulated in androgen-independent prostate cancer cell lines. The level of antioxidant enzymes, catalase, MnSOD and CuZnSOD are up-regulated in prostate cancer cell lines. Furthermore, it was observed that the activity of mGPDH is significantly higher in liver tissues from all mice with cancer compared to liver tissues from control mice. These data suggest that the up-regulation of mGPDH, due to a highly glycolytic environment, contributes to the overall increase in ROS generation and may result in the progression of the cancer.     

Elucidation of the effects of lipoperoxidation on the mitochondrial electron transport chain using yeast mitochondria with manipulated fatty acid content

Lipoperoxidative damage to the respiratory chain proteins may account for disruption in mitochondrial electron transport chain (ETC) function and could lead to an augment in the production of reactive oxygen species (ROS). To test this hypothesis, we investigated the effects of lipoperoxidation on ETC function and cytochromes spectra of Saccharomyces cerevisiae mitochondria. We compared the effects of Fe²⁺ treatment on mitochondria isolated from yeast with native (lipoperoxidation-resistant) and modified (lipoperoxidation-sensitive) fatty acid composition. Augmented sensitivity to oxidative stress was observed in the complex III-complex IV segment of the ETC. Lipoperoxidation did not alter the cytochromes content. Under lipoperoxidative conditions, cytochrome c reduction by succinate was almost totally eliminated by superoxide dismutase and stigmatellin. Our results suggest that lipoperoxidation impairs electron transfer mainly at cytochrome b in complex III, which leads to increased resistance to antimycin A and ROS generation due to an electron leak at the level of the Q_O site of complex III.