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.     

Diphenyleneiodonium acutely inhibits reactive oxygen species production by mitochondrial complex I during reverse, but not forward electron transport

We investigated the effects of diphenyleneiodonium (DPI) on superoxide production by complex I in mitochondria isolated from rat skeletal muscle. Superoxide production was measured indirectly as hydrogen peroxide production. In a conventional medium containing chloride, DPI strongly inhibited superoxide production by complex I driven by reverse electron transport from succinate. In principle, this inhibition could be explained by an observed decrease in the mitochondrial pH gradient caused by the known chloride-hydroxide antiport activity of DPI. In a medium containing gluconate instead of chloride, DPI did not affect the pH gradient. In this gluconate medium, DPI still inhibited superoxide production driven by reverse electron transport, showing that the inhibition of superoxide production was not dependent on changes in the pH gradient. It had no effect on superoxide production during forward electron transport from NAD-linked substrates in the presence of rotenone (to maximise superoxide production from the flavin of complex I) or antimycin (to maximise superoxide production from complex III), suggesting that the effects of DPI were not through inhibition of the flavin. We conclude that DPI has the novel and potentially very useful ability to prevent superoxide production from the site in complex I that is active during reverse electron transport, without affecting superoxide production during forward electron transport.     

Diphenyleneiodonium acutely inhibits reactive oxygen species production by mitochondrial complex I during reverse, but not forward electron transport

We investigated the effects of diphenyleneiodonium (DPI) on superoxide production by complex I in mitochondria isolated from rat skeletal muscle. Superoxide production was measured indirectly as hydrogen peroxide production. In a conventional medium containing chloride, DPI strongly inhibited superoxide production by complex I driven by reverse electron transport from succinate. In principle, this inhibition could be explained by an observed decrease in the mitochondrial pH gradient caused by the known chloride-hydroxide antiport activity of DPI. In a medium containing gluconate instead of chloride, DPI did not affect the pH gradient. In this gluconate medium, DPI still inhibited superoxide production driven by reverse electron transport, showing that the inhibition of superoxide production was not dependent on changes in the pH gradient. It had no effect on superoxide production during forward electron transport from NAD-linked substrates in the presence of rotenone (to maximise superoxide production from the flavin of complex I) or antimycin (to maximise superoxide production from complex III), suggesting that the effects of DPI were not through inhibition of the flavin. We conclude that DPI has the novel and potentially very useful ability to prevent superoxide production from the site in complex I that is active during reverse electron transport, without affecting superoxide production during forward electron transport.     

Reactive oxygen species and oxidative stress

Oxidative stress is defined as an imbalance between the production of reactive oxygen species (ROS) and the antioxidant capacity of the cell. For long, ROS have been considered as harmful by-products of the normal aerobic metabolism process of the mitochondria, implicated in a large variety of diseases. But there are now growing evidences that controlled ROS production also play physiological roles especially in regulating cell redox homeostasis and cell signaling. Biological ROS effects are now well documented. Data show that living organisms have not only adapted themselves to coexist with free radicals but have also developed mechanisms to use them advantageously. However their main sources and mechanisms of action remain poorly described. This review focuses on the main properties of ROS and their paradoxical effects.     

Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production

Inhibition of mitochondrial respiratory chain complex I by rotenone had been found to induce cell death in a variety of cells. However, the mechanism is still elusive. Because reactive oxygen species (ROS) play an important role in apoptosis and inhibition of mitochondrial respiratory chain complex I by rotenone was thought to be able to elevate mitochondrial ROS production, we investigated the relationship between rotenone-induced apoptosis and mitochondrial reactive oxygen species. Rotenone was able to induce mitochondrial complex I substrate-supported mitochondrial ROS production both in isolated mitochondria from HL-60 cells as well as in cultured cells. Rotenone-induced apoptosis was confirmed by DNA fragmentation, cytochrome c release, and caspase 3 activity. A quantitative correlation between rotenone-induced apoptosis and rotenone-induced mitochondrial ROS production was identified. Rotenone-induced apoptosis was inhibited by treatment with antioxidants (glutathione, N-acetylcysteine, and vitamin C). The role of rotenone-induced mitochondrial ROS in apoptosis was also confirmed by the finding that HT1080 cells overexpressing magnesium superoxide dismutase were more resistant to rotenone-induced apoptosis than control cells. These results suggest that rotenone is able to induce apoptosis via enhancing the amount of mitochondrial reactive oxygen species production.     

Generation of reactive oxygen species by the mitochondrial electron transport chain

Generation of reactive oxygen species (ROS) by the mitochondrial electron transport chain (ETC), which is composed of four multiprotein complexes named complex I-IV, is believed to be important in the aging process and in the pathogenesis of neurodegenerative diseases such as Parkinson's disease. Previous studies have identified the ubiquinone of complex III and an unknown component of complex I as the major sites of ROS generation. Here we show that the physiologically relevant ROS generation supported by the complex II substrate succinate occurs at the flavin mononucleotide group (FMN) of complex I through reversed electron transfer, not at the ubiquinone of complex III as commonly believed. Indirect evidence indicates that the unknown ROS-generating site within complex I is also likely to be the FMN group. It is therefore suggested that the major physiologically and pathologically relevant ROS-generating site in mitochondria is limited to the FMN group of complex I. These new insights clarify an elusive target for intervening mitochondrial ROS-related processes or diseases.     

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.     

Reactive oxygen species differentially affect T cell receptor-signaling pathways

Oxidative stress plays an important role in the induction of T lymphocyte hyporesponsiveness observed in several human pathologies including cancer, rheumatoid arthritis, leprosy, and AIDS. To investigate the molecular basis of oxidative stress-induced T cell hyporesponsiveness, we have developed an in vitro system in which T lymphocytes are rendered hyporesponsive by co-culture with oxygen radical-producing activated neutrophils. We have observed a direct correlation between the level of T cell hyporesponsiveness induced and the concentration of reactive oxygen species produced. Moreover, induction of T cell hyporesponsiveness is blocked by addition of N-acetyl cysteine, Mn(III)tetrakis(4-benzoic acid)porphyrin chloride, and catalase, confirming the critical role of oxidative stress in this system. The pattern of tyrosine-phosphorylated proteins was profoundly altered in hyporesponsive as compared with normal T cells. In hyporesponsive T cells, T cell receptor (TCR) ligation no longer induced phospholipase C-gamma1 activation and caused reduced Ca(2+) flux. In contrast, despite increased levels of ERK1/2 phosphorylation, TCR-dependent activation of mitogen-activated protein kinase ERK1/2 was unaltered in hyporesponsive T lymphocytes. A late TCR-signaling event such as caspase 3 activation was as well unaffected in hyporesponsive T lymphocytes. Our data indicate that TCR-signaling pathways are differentially affected by physiological levels of oxidative stress and would suggest that although "hyporesponsive" T cells have lost certain effector functions, they may have maintained or gained others.     

Reactive oxygen species production by forward and reverse electron fluxes in the mitochondrial respiratory chain

Reactive oxygen species (ROS) produced in the mitochondrial respiratory chain (RC) are primary signals that modulate cellular adaptation to environment, and are also destructive factors that damage cells under the conditions of hypoxia/reoxygenation relevant for various systemic diseases or transplantation. The important role of ROS in cell survival requires detailed investigation of mechanism and determinants of ROS production. To perform such an investigation we extended our rule-based model of complex III in order to account for electron transport in the whole RC coupled to proton translocation, transmembrane electrochemical potential generation, TCA cycle reactions, and substrate transport to mitochondria. It fits respiratory electron fluxes measured in rat brain mitochondria fueled by succinate or pyruvate and malate, and the dynamics of NAD(+) reduction by reverse electron transport from succinate through complex I. The fitting of measured characteristics gave an insight into the mechanism of underlying processes governing the formation of free radicals that can transfer an unpaired electron to oxygen-producing superoxide and thus can initiate the generation of ROS. Our analysis revealed an association of ROS production with levels of specific radicals of individual electron transporters and their combinations in species of complexes I and III. It was found that the phenomenon of bistability, revealed previously as a property of complex III, remains valid for the whole RC. The conditions for switching to a state with a high content of free radicals in complex III were predicted based on theoretical analysis and were confirmed experimentally. These findings provide a new insight into the mechanisms of ROS production in RC.     

The effect of reactive oxygen species generated from the mitochondrial electron transport chain on the cytochrome c oxidase activity and on the cardiolipin content in bovine heart submitochondrial particles

The effect of reactive oxygen species (ROS), produced by the mitochondrial respiratory chain, on the activity of cytochrome c oxidase and on the cardiolipin content in bovine heart submitochondrial particles (SMP) was studied. ROS were produced by treatment of succinate-respiring SMP with antimycin A. This treatment resulted in a large production of superoxide anion, measured by epinephrine method, which was blocked by superoxide dismutase (SOD). Exposure of SMP to mitochondrial mediated ROS generation, led to a marked loss of cytochrome c oxidase activity and to a parallel loss of cardiolipin content. Both these effects were completely abolished by SOD+catalase. Added cardiolipin was able to almost completely restore the ROS-induced loss of cytochrome c oxidase activity. No restoration was obtained with peroxidized cardiolipin. These results demonstrate that mitochondrial mediated ROS generation affects the activity of cytochrome c oxidase via peroxidation of cardiolipin which is needed for the optimal functioning of this enzyme complex. These results may prove useful in probing molecular mechanism of ROS-induced peroxidative damage to mitochondria which have been proposed to contribute to aging, ischemia/reperfusion and chronic degenerative diseases.     

Reactive oxygen species regulate autophagy through redox-sensitive proteases

Starvation induces autophagy through a signal transduction pathway that is not fully understood. In a recent issue of The EMBO Journal, Scherz-Shouval and colleagues (Scherz-Shouval et al., 2007) show that reactive oxygen species (ROS) occurring during starvation serve as signaling molecules that initiate autophagy.     

Reaction of complex I of the mitochondrial electron transport chain with artificial oxidizers

The steady-state kinetics of oxidation of the mitochondrial NADH: ubiquinone oxidoreductase (complex I, EC 1.6.99.3) by artificial electron acceptors--p-quinones and inorganic complexes has been investigated. A limiting stage in the NADH: ferricyanide reductase reaction is a reductive half-reaction. Ferricyanide interacts with negative-charged protein groups taking part in the NADH binding. The rate constants of the quinone reduction by complex I vary from 1.10(6) to 4.10(3) M-1s-1. The NADH, NAD+ and ADP-ribose inhibition data indicate that oxidizers in the rotenono-insensitive reaction interact with the redox centre near the NAD+/NADH binding site, most probably with FMN.     

Mitochondrial dysfunction in rat brain with aging Involvement of complex I, reactive oxygen species and cardiolipin

Reactive oxygen species (ROS) are considered a key factor in brain aging process. Mitochondrial respiration is an important site of ROS production and hence a potential contributor to brain functional changes with aging. In this study we examined the effect of aging on complex I activity, oxygen consumption, ROS production and phospholipid composition in rat brain mitochondria. The activity of complex I was reduced by 30% in brain mitochondria from 24 months aged rats relative to young animals. These changes in complex I activity were associated with parallel changes in state 3 respiration. H(2)O(2) generation was significantly increased in mitochondria isolated from aged rats. The mitochondrial content of cardiolipin, a phospholipid required for optimal activity of complex I, decreased by 31% as function of aging, while there was a significant increase in the level of peroxidized cardiolipin. The age-related decrease in complex I activity in brain mitochondria could be reversed by exogenously added cardiolipin. This effect of cardiolipin could not be replaced by other phospholipids. It is proposed that aging causes brain mitochondrial complex I dysfunction which can be attributed to ROS-induced cardiolipin oxidation. These findings may prove useful in elucidating the mechanism underlying mitochondrial dysfunction associated with brain aging.     

TNF-induced mitochondrial damage: a link between mitochondrial complex I activity and left ventricular dysfunction

Mitochondrial damage is implicated in the progression of cardiac disease. Considerable evidence suggests that proinflammatory cytokines induce oxidative stress and contribute to cardiac dysfunction. This study was conducted to determine whether a TNF-induced increase in superoxide (O₂^?) contributes to mitochondrial damage in the left ventricle (LV) by impairing respiratory complex I activity. We employed an electron paramagnetic resonance (EPR) method to measure O₂^? and oxygen consumption in mitochondrial respiratory complexes, using an oxygen label. Adult male Sprague–Dawley rats were divided into four groups: control, TNF treatment (ip), TNF+ apocynin (APO; 200 μmol/kg bw, orally), and TNF+ Tempol (Temp; 300 μmol/kg bw, orally). TNF was injected daily for 5 days. Rats were sacrificed, LV tissue was collected, and mitochondria were isolated for EPR studies. Total LV ROS production was significantly higher in TNF animals than in controls; APO or Temp treatment ameliorated TNF-induced LV ROS production. Total mitochondrial ROS production was significantly higher in the TNF and TNF+ APO groups than in the control and TNF+ Temp groups. These findings suggest that TNF alters the cellular redox state, reduces the expression of four complex I subunits by increasing mitochondrial O₂^? production and depleting ATP synthesis, and decreases oxygen consumption, thereby resulting in mitochondrial damage and leading to LV dysfunction.     

Arachidonic acid interaction with the mitochondrial electron transport chain promotes reactive oxygen species generation.

A study has been carried out on the interaction of arachidonic acid and other long chain free fatty acids with bovine heart mitochondria. It is shown that arachidonic acid causes an uncoupling effect under state 4 respiration of intact mitochondria as well as a marked inhibition of uncoupled respiration. While, under our conditions, the uncoupling effect is independent of the fatty acid species considered, the inhibition is stronger for unsaturated acids. Experiments carried out with mitochondrial particles indicated that the arachidonic acid dependent decrease of the respiratory activity is caused by a selective inhibition of Complex I and III. It is also shown that arachidonic acid causes a remarkable increase of hydrogen peroxide production when added to mitochondria respiring with either pyruvate+malate or succinate as substrate. The production of reactive oxygen species (ROS) at the coupling site II was almost double than that at site I. The results obtained are discussed with regard to the impairment of the mitochondrial respiratory activity as occurring during the heart ischemia/reperfusion process.     

Reactive oxygen species induce cardiomyocyte apoptosis partly through TNF-alpha

Many studies have indicated that oxidative stress induces apoptosis in cardiomyocytes, but its mechanism remains unknown. We examined whether tumor necrosis factor-alpha (TNF-alpha) is involved in oxidative stress-induced cardiomyocyte apoptosis. Pretreatment with anti-TNF-alpha antibody significantly decreased the number of H(2)O(2)-induced TUNEL-positive cardiomyocytes. Expression of TNF-alpha gene was upregulated by H(2)O(2), and H(2)O(2) mildly but significantly increased the concentration of TNF-alpha in the culture medium. Although neither low dose of H(2)O(2) nor TNF-alpha induced apoptosis, stimulation with H(2)O(2) and TNF-alpha synergistically increased apoptosis. These results suggest that oxidative stress induces apoptosis of cardiac myocytes partly through TNF-alpha.     

Depolarization and cardiolipin depletion in aged rat brain mitochondria: relationship with oxidative stress and electron transport chain activity

A noticeable loss of cardiolipin, a significant accumulation of fluorescent products of lipid peroxidation and an increased ability to produce reactive oxygen species in vitro are characteristics of aged rat brain mitochondria, as has been demonstrated in this study. In contrast mitochondrial electron transport chain activity is not significantly compromised except a marginal decline in complex IV activity in aged rat brain. On the other hand, a striking loss of mitochondrial membrane potential occurs in brain mitochondria during aging, which may be attributed to peroxidative membrane damage in this condition. Such mitochondrial dysfunctions as reported here may lead to uncoupling of oxidative phosphorylation, ATP depletion and activation of apoptotic cascade in aged rat brain.     

Brain mitochondrial complex I inactivation by oxidative modification

In vitro oxidation of the brain mitochondrial complex I by the hydroxyl radical generating system ascorbate/Fe(III)/O(2) has been carried out. Complex I inactivation, by oxidation, has been studied using a method based on the resolution of proteins by blue native polyacrylamide gel electrophoresis (BN-PAGE), followed by total protein quantification by staining with Coomassie brilliant blue, in-gel activity quantification, and quantification of oxidized proteins by labelling with DIG-hydrazide and immunodetection with an anti-DIG-AP. Quantification was carried out by densitometry procedure. Our results show that oxidation is a continuous process, increasing rapidly at the beginning, reaching a plateau after 8 h of incubation. There is practically no inactivation until a threshold value of damage is reached. Below this, the complex activity is resistant to the aggression of oxygen-reactive substances and free radicals, but once the threshold value is passed, activity is lost rapidly.     

Mitochondrial complex I dysfunction in rat heart with aging: critical role of reactive oxygen species and cardiolipin

Reactive oxygen species (ROS) are considered a key factor in the heart aging process. Mitochondrial respiration is an important site of ROS generation and a potential contributor to heart functional changes with aging. We have examined the effects of aging on various parameters related to mitochondrial bioenergetics in rat heart, such as complex I activity, oxygen consumption, membrane potential, ROS production, and cardiolipin content and oxidation. A loss in complex I activity, state 3 respiration, and membrane potential was found in mitochondria with aging. The capacity of mitochondria to produce H(2)O(2) was significantly increased in aged rats. The mitochondrial content of cardiolipin, a phospholipid required for optimal activity of complex I, significantly decreased as a function of aging, whereas there was a significant increase in the level of oxidized cardiolipin. The lower complex I activity in mitochondria from aged rats could be almost completely restored to the level of young heart by exogenously added cardiolipin, but not by other phospholipids nor by peroxidized cardiolipin. It is proposed that aging causes heart mitochondrial complex I deficiency, which can be attributed to ROS-induced cardiolipin peroxidation. These results may prove useful in elucidating the mechanism underlying mitochondrial dysfunction associated with heart aging.