A simple assay for inhibitors of mycobacterial oxidative phosphorylation.

Oxidative phosphorylation, the combined activities of the electron transport chain (ETC) and ATP synthase, has emerged as a valuable target for antibiotics to treat infection with Mycobacterium tuberculosis and related pathogens. In oxidative phosphorylation, the ETC establishes a transmembrane electrochemical proton gradient that powers ATP synthesis. Monitoring oxidative phosphorylation with luciferase-based detection of ATP synthesis or measurement of oxygen consumption can be technically challenging and expensive. These limitations reduce the utility of these methods for characterization of mycobacterial oxidative phosphorylation inhibitors. Here, we show that fluorescence-based measurement of acidification of inverted membrane vesicles (IMVs) can detect and distinguish between inhibition of the ETC, inhibition of ATP synthase, and nonspecific membrane uncoupling. In this assay, IMVs from Mycobacterium smegmatis are acidified either through the activity of the ETC or ATP synthase, the latter modified genetically to allow it to serve as an ATP-driven proton pump. Acidification is monitored by fluorescence from 9-amino-6-chloro-2-methoxyacridine, which accumulates and quenches in acidified IMVs. Nonspecific membrane uncouplers prevent both succinate- and ATP-driven IMV acidification. In contrast, the ETC Complex III2IV2 inhibitor telacebec (Q203) prevents succinate-driven acidification but not ATP-driven acidification, and the ATP synthase inhibitor bedaquiline prevents ATP-driven acidification but not succinate-driven acidification. We use the assay to show that, as proposed previously, lansoprazole sulfide is an inhibitor of Complex III2IV2, whereas thioridazine uncouples the mycobacterial membrane nonspecifically. Overall, the assay is simple, low cost, and scalable, which will make it useful for identifying and characterizing new mycobacterial oxidative phosphorylation inhibitors.

Potent inhibition of tumour cell proliferation and immunoregulatory function by mitochondria-targeted atovaquone.

The FDA-approved prophylactic antimalarial drug atovaquone (ATO) recently was repurposed as an antitumor drug. Studies show that ATO exerts a profound antiproliferative effect in several cancer cells, including breast, ovarian, and glioma. Analogous to the mechanism of action proposed in parasites, ATO inhibits mitochondrial complex III and cell respiration. To enhance the chemotherapeutic efficacy and oxidative phosphorylation inhibition, we developed a mitochondria-targeted triphenylphosphonium-conjugated ATO with varying alkyl side chains (Mito4-ATO, Mito10-ATO, Mito12-ATO, and Mito16-ATO). Results show, for the first time, that triphenylphosphonium-conjugated ATO potently enhanced the antiproliferative effect of ATO in cancer cells and, depending upon the alkyl chain length, the molecular target of inhibition changes from mitochondrial complex III to complex I. Mito4-ATO and Mito10-ATO inhibit both pyruvate/malate-dependent complex I and duroquinol-dependent complex III-induced oxygen consumption whereas Mito12-ATO and Mito16-ATO inhibit only complex I-induced oxygen consumption. Mitochondrial target shifting may have immunoregulatory implications.

The potential of respiration inhibition as a new approach to combat human fungal pathogens.

The respiratory chain has been proposed as an attractive target for the development of new therapies to tackle human fungal pathogens. This arises from the presence of fungal-specific electron transport chain components and links between respiration and the control of virulence traits in several pathogenic species. However, as the physiological roles of mitochondria remain largely undetermined with respect to pathogenesis, its value as a potential new drug target remains to be determined. The use of respiration inhibitors as fungicides is well developed but has been hampered by the emergence of rapid resistance to current inhibitors. In addition, recent data suggest that adaptation of the human fungal pathogen, Candida albicans, to respiration inhibitors can enhance virulence traits such as yeast-to-hypha transition and cell wall organisation. We conclude that although respiration holds promise as a target for the development of new therapies to treat human fungal infections, we require a more detailed understanding of the role that mitochondria play in stress adaption and virulence.

Repurposing of an old drug: In vitro and in vivo efficacies of buparvaquone against Echinococcus multilocularis.

The metacestode stage of the fox tapeworm Echinococcus multilocularis causes the lethal disease alveolar echinococcosis. Current chemotherapeutic treatment options are based on benzimidazoles (albendazole and mebendazole), which are insufficient and hence alternative drugs are needed. In this study, we screened the 400 compounds of the Medicines for Malaria Venture (MMV) Pathogen Box against E. multilocularis metacestodes. For the screen, we employed the phosphoglucose isomerase (PGI) assay which assesses drug-induced damage on metacestodes, and identified ten new compounds with activity against the parasite. The anti-theilerial drug MMV689480 (buparvaquone) and MMV671636 (ELQ-400) were the most promising compounds, with an IC50 of 2.87 µM and 0.02 µM respectively against in vitro cultured E. multilocularis metacestodes. Both drugs suggested a therapeutic window based on their cytotoxicity against mammalian cells. Transmission electron microscopy revealed that treatment with buparvaquone impaired parasite mitochondria early on and additional tests showed that buparvaquone had a reduced activity under anaerobic conditions. Furthermore, we established a system to assess mitochondrial respiration in isolated E. multilocularis cells in real time using the Seahorse XFp Analyzer and demonstrated inhibition of the cytochrome bc1 complex by buparvaquone. Mice with secondary alveolar echinococcosis were treated with buparvaquone (100 mg/kg per dose, three doses per week, four weeks of treatment), but the drug failed to reduce the parasite burden in vivo. Future studies will reveal whether improved formulations of buparvaquone could increase its effectivity.

Inconsistent responses of liver mitochondria metabolism and standard metabolism in Silurus meridionalis when exposed to waterborne cadmium.

We investigated the standard metabolic rate and liver mitochondria metabolism of the southern catfish when exposed to waterborne cadmium. Juvenile southern catfish were exposed to waterborne cadmium concentrations (0, 62.5, 125, 250 and 500 µg/L, respectively) for 8 weeks, and the final body mass, the standard metabolic rate, the state III respiration rate, the activity of cytochrome C oxidase (CCO) of liver mitochondria, and the hepatosomatic index (HSI) were determined. The results showed that the 62.5 µg/L, 125 µg/L, and 250 µg/L experiment groups had a significantly higher standard metabolic rate than that of the control group. Standard metabolic rate in the 500 µg/L experiment group did not differ from the control group. State III respiration rate of liver mitochondria decreased with an increase in cadmium concentration. The 125 µg/L, 250 µg/L, and 500 µg/L experiment groups had a significantly lower state III respiration rate than that of the control group. The activity of CCO in the 500 µg/L experiment group was significantly lower than that of the control group. These results suggest that at low cadmium concentrations, the southern catfish could continuously improve the standard metabolism to provide extra energy in response to the cadmium stress. Cadmium exposures caused damage to the structure and function of liver mitochondria and decreased the activity of mitochondria enzymes, which results in a decrease in the energy of the liver metabolism. The adjustment of the metabolism of liver mitochondria in southern catfish was inconsistent with the adjustment of individual standard metabolism.

Chemicals or mutations that target mitochondrial translation can rescue the respiratory deficiency of yeast bcs1 mutants.

Bcs1p is a chaperone that is required for the incorporation of the Rieske subunit within complex III of the mitochondrial respiratory chain. Mutations in the human gene BCS1L (BCS1-like) are the most frequent nuclear mutations resulting in complex III-related pathologies. In yeast, the mimicking of some pathogenic mutations causes a respiratory deficiency. We have screened chemical libraries and found that two antibiotics, pentamidine and clarithromycin, can compensate two bcs1 point mutations in yeast, one of which is the equivalent of a mutation found in a human patient. As both antibiotics target the large mtrRNA of the mitoribosome, we focused our analysis on mitochondrial translation. We found that the absence of non-essential translation factors Rrf1 or Mif3, which act at the recycling/initiation steps, also compensates for the respiratory deficiency of yeast bcs1 mutations. At compensating concentrations, both antibiotics, as well as the absence of Rrf1, cause an imbalanced synthesis of respiratory subunits which impairs the assembly of the respiratory complexes and especially that of complex IV. Finally, we show that pentamidine also decreases the assembly of complex I in nematode mitochondria. It is well known that complexes III and IV exist within the mitochondrial inner membrane as supramolecular complexes III2/IV in yeast or I/III2/IV in higher eukaryotes. Therefore, we propose that the changes in mitochondrial translation caused by the drugs or by the absence of translation factors, can compensate for bcs1 mutations by modifying the equilibrium between illegitimate, and thus inactive, and active supercomplexes.

The antileishmanial activity of xanthohumol is mediated by mitochondrial inhibition.

Xanthohumol (Xan) is a natural constituent of human nutrition. Little is known about its actions on leishmanial parasites and their mitochondria as putative target. Therefore, we determined the antileishmanial activity of Xan and resveratrol (Res, as alternative compound with antileishmanial activity) with respect to mitochondria in Leishmania amazonensis promastigotes/amastigotes (LaP/LaA) in comparison with their activity in peritoneal macrophages from mouse (PMM) and macrophage cell line J774A.1 (J774). Mechanistic studies were conducted in Leishmania tarentolae promastigotes (LtP) and mitochondrial fractions isolated from LtP. Xan and Res demonstrated antileishmanial activity in LaA [half inhibitory concentration (IC50): Xan 7 µ m, Res 14 µ m]; while they had less influence on the viability of PMM (IC50: Xan 70 µ m, Res >438 µ m). In contrast to Res, Xan strongly inhibited oxygen consumption in Leishmania (LtP) but not in J774 cells. This was based on the inhibition of the mitochondrial electron transfer complex II/III by Xan, which was less pronounced with Res. Neither Xan nor Res increased mitochondrial superoxide release in LtP, while both decreased the mitochondrial membrane potential in LtP. Bioenergetic studies showed that LtP mitochondria have no spare respiratory capacity in contrast to mitochondria in J774 cells and can therefore much less adapt to stress by mitochondrial inhibitors, such as Xan. These data show that Xan may have antileishmanial activity, which is mediated by mitochondrial inhibition.

Increased myocardial vulnerability to ischemia-reperfusion injury in the presence of left ventricular hypertrophy.

OBJECTIVE: Despite its high prevalence among patients suffering myocardial infarction, the significance of left ventricle hypertrophy for infarct size is not known. We asked whether infarct size might be increased by this condition, and whether any such increase might be associated with an increased mitochondrial damage following coronary occlusion. METHODS: Occlusion of the left descending artery in isolated, perfused hearts of SHR-SP (spontaneously hypertensive rat stroke-prone) (left ventricular hypertrophy) or Wistar-Kyoto (WKY) (control) rats was used, followed by reperfusion with or without exendin-4 (Exe-4), a glucagon-like peptide-1 receptor agonist. Infarct size relative to area-at-risk was determined. Separately, mitochondria were isolated after global ischemia. Activities of complexes III and IV and amounts of selected complex subunits and cytochromes a, b, c, and c1 were determined. RESULTS: Infarct size (ischemia 35  min and 120  min reperfusion) was 65.8% (±3.3%) and 37.1% (±3.4%) in the SHR-SP and WKY hearts, respectively (P < 0.05). Exe-4 significantly decreased infarct size and hypercontracture in WKY, but not in SHR-SP, hearts. After ischemia 15  min in SHR-SP hearts, Exe-4 reduced the infarct (26.6%, ±3.8% to 9.3% ± 1.5%; P < 0.05). Mitochondria from postischemic SHR-SP hearts showed a reduction of complex III (368.1 ± 37.5 to 175.8 ± 23.0  nmoles/min × mg; P < 0.05) and complex IV (14.4 ± 0.22 to 5.8 ± 0.8 1/s × mg; P < 0.05) activities and decreased amounts of cytochromes a, b, and c. CONCLUSION: Hearts from hypertensive (SHR-SP) rats with left ventricle hypertrophy appeared more vulnerable to ischemia-reperfusion injury, as supported by a more profound infarct development and an earlier loss of postconditioning by Exe-4. Mitochondrial complexes III and IV were identified among possible loci of this increased, hypertrophy-associated vulnerability.

Lansoprazole is an antituberculous prodrug targeting cytochrome bc1.

Better antibiotics capable of killing multi-drug-resistant Mycobacterium tuberculosis are urgently needed. Despite extensive drug discovery efforts, only a few promising candidates are on the horizon and alternative screening protocols are required. Here, by testing a panel of FDA-approved drugs in a host cell-based assay, we show that the blockbuster drug lansoprazole (Prevacid), a gastric proton-pump inhibitor, has intracellular activity against M. tuberculosis. Ex vivo pharmacokinetics and target identification studies reveal that lansoprazole kills M. tuberculosis by targeting its cytochrome bc1 complex through intracellular sulfoxide reduction to lansoprazole sulfide. This novel class of cytochrome bc1 inhibitors is highly active against drug-resistant clinical isolates and spares the human H(+)K(+)-ATPase thus providing excellent opportunities for targeting the major pathogen M. tuberculosis. Our finding provides proof of concept for hit expansion by metabolic activation, a powerful tool for antibiotic screens.

Saccharomyces cerevisiae-based mutational analysis of the bc1 complex Qo site residue 279 to study the trade-off between atovaquone resistance and function.

The bc1 complex is central to mitochondrial bioenergetics and the target of the antimalarial drug atovaquone that binds in the quinol oxidation (Qo) site of the complex. Structural analysis has shown that the Qo site residue Y279 (Y268 in Plasmodium falciparum) is key for atovaquone binding. Consequently, atovaquone resistance can be acquired by mutation of that residue. In addition to the probability of amino acid substitution, the level of atovaquone resistance and the loss of bc1 complex activity that are associated with the novel amino acid would restrict the nature of resistance-driven mutations occurring on atovaquone exposure in native parasite populations. Using the yeast model, we characterized the effect of all the amino acid replacements resulting from a single nucleotide substitution at codon 279: Y279C, Y279D, Y279F, Y279H, Y279N, and Y279S (Y279C, D, F, H, N, and S). Two residue changes that required a double nucleotide substitution, Y279A and W, were added to the series. We found that mutations Y279A, C, and S conferred high atovaquone resistance but decreased the catalytic activity. Y279F had wild-type enzymatic activity and sensitivity to atovaquone, while the other substitutions caused a dramatic respiratory defect. The results obtained with the yeast model were examined in regard to atomic structure and compared to the reported data on the evolution of acquired atovaquone resistance in P. falciparum.

Effects of tramadol, clonazepam, and their combination on brain mitochondrial complexes.

The present study is an unsubstantiated qualitative assessment of the abused drugs-tramadol and clonazepam. The aim of this study is to evaluate whether the effects of tramadol, clonazepam, and their combination on mitochondrial electron transport chain (ETC) complexes were influential at therapeutic or at progressively increasing doses. The study comprised of a total of 70 healthy male rats, aged 3 months. According to the drug intake regimen, animals were divided into seven groups: control, tramadol therapeutic, clonazepam therapeutic, combination therapeutic, tramadol abuse, clonazepam abuse, and combination abuse group. At the end of the experiment, brain mitochondrial ETC complexes (I, II, III, and IV) were evaluated. Histopathological examinations were also performed on brain tissues. The results showed that groups that received tramadol (therapeutic and abuse) suffered from weight loss. Tramadol abuse group and combination abuse group showed significant decrease in the activities of I, III, and IV complexes but not in the activity of complex II. In conclusion, tramadol but not clonazepam has been found to partially inhibit the activities of respiratory chain complexes I, III, and IV but not the activity of complex II and such inhibition occurred only at doses that exceeded the maximum recommended adult human daily therapeutic doses. This result explains the clinical and histopathological effects of tramadol, such as seizures and red neurons (marker for apoptosis), respectively.

Heptachlor induced mitochondria-mediated cell death via impairing electron transport chain complex III.

Environmental toxins like pesticides have been implicated in the pathogenesis of Parkinson's disease (PD). Epidemiological studies suggested that exposures to organochlorine pesticides have an association with an increased PD risk. In the present study, we examined the mechanism of toxicity induced by an organochlorine pesticide heptachlor. In a human dopaminergic neuroblastoma SH-SY5Y cells, heptachlor induced both morphological and functional damages in mitochondria. Interestingly, the compound inhibited mitochondrial electron transport chain complex III activity. Rapid generation of reactive oxygen species and the activation of Bax were then detected. Subsequently, mitochondria-mediated, caspase-dependent apoptosis followed. Our results raise a possibility that an organochlorine pesticide heptachlor can act as a neurotoxicant associated with PD.

A rapid in vivo colorimetric library screen for inhibitors of microbial respiration.

A number of fungicides that target the respiratory chain enzymes complexes II and III are used in agriculture. They are active against a large range of phytopathogens. Unfortunately, the evolution of fungicide resistance has quickly become a major issue. Resistance is often caused by mutations in the inhibitor binding domains of the complexes, and new molecules are required that are able to bypass such resistance mutations. We report here on a rapid in vivo high-throughput method, using yeast and the redox dye TTC to screen chemical libraries and identify inhibitors of respiratory function. We applied that screening process, followed by a series of tests, to a diverse library of 4,640 molecules and identified a weak inhibitor of complex III without toxic effect on the cell. Interestingly, that drug (D12) is fully active against the mutant enzyme harboring the G143A mutation that confers a high level of resistance toward most of the fungicides targeting complex III but is not active against bovine complex III. Using a collection of yeast strains harboring mutations in the inhibitor binding sites (Q(o) and Q(i) sites), we showed that D12 targeted the Q(o) site and that its inhibitory activity was weakened by the mutation L275F. A phenylalanine is naturally present at position 275 in mammalian complex III, which could explain the differential sensitivity toward D12. The molecule is not structurally related to commercial inhibitors of complex III and could potentially be used as a lead compound for the development of antimicrobial agents.

2-amino-nonyl-6-methoxyl-tetralin muriate activity against Candida albicans augments endogenous reactive oxygen species production --a microarray analysis study.

Candida infections have become an increasingly significant problem, mainly because of the widespread nature of Candida and drug resistance. There is an urgent need to develop new classes of drugs for the treatment of opportunistic Candida infections, especially in medically complex patients. Previous studies have confirmed that 2-amino-nonyl-6-methoxyl-tetralin muriate (10b) possesses powerful antifungal activity in vitro against Candia albicans. To clarify the underlying action mechanism, an oligonucleotide microarray study was performed in C. albicans SC5314 without and with 10b treatment. The analytical results showed that energy metabolism-related genes, including glycolysis-related genes (PFK1, CDC19 and HXK2), fermentation-related genes (PDC11, ALD5 and ADH1) and respiratory electron transport chain-related genes (CBP3, COR1 and QCR8), were downregulated significantly. Functional analysis revealed that 10b treatment increased the generation of endogenous reactive oxygen species, and decreased mitochondrial membrane potential, ubiquinone-cytochrome c reductase (complex III) activity and intracellular ATP levels in C. albicans SC5314. Also, addition of the antioxidant ascorbic acid reduced the antifungal activity of 10b significantly. These results suggest that mitochondrial aerobic respiration shift and endogenous reactive oxygen species augmentation might contribute to the antifungal activity of 10b against C. albicans. This information may prove to be useful for the development of new strategies to treat Candida infections.

Tafenoquine, an antiplasmodial 8-aminoquinoline, targets leishmania respiratory complex III and induces apoptosis.

Tafenoquine (TFQ), an 8-aminoquinoline analogue of primaquine, which is currently under clinical trial (phase IIb/III) for the treatment and prevention of malaria, may represent an alternative treatment for leishmaniasis. In this work, we have studied the mechanism of action of TFQ against Leishmania parasites. TFQ impaired the overall bioenergetic metabolism of Leishmania promastigotes, causing a rapid drop in intracellular ATP levels without affecting plasma membrane permeability. TFQ induced mitochondrial dysfunction through the inhibition of cytochrome c reductase (respiratory complex III) with a decrease in the oxygen consumption rate and depolarization of mitochondrial membrane potential. This was accompanied by ROS production, elevation of intracellular Ca(2+) levels and concomitant nuclear DNA fragmentation. We conclude that TFQ targets Leishmania mitochondria, leading to an apoptosis-like death process.

Propamidine decreas mitochondrial complex III activity of Botrytis cinerea.

Propamidine, an aromatic diamidine compound, is widely used as an antimicrobial agent. To uncover its mechanism on pathogenetic fungi, Botrytis cinerea as an object was used to investigate effects of propamidine in this paper. The transmission electron microscope results showed that the mitochondrial membranes were collapsed after propamidine treatment, followed that mitochondria were disrupted. Inhibition of whole-cell and mitochondrial respiration by propamidine suggested that Propamidine is most likely an inhibitor of electron transport within Botrytis cinerea mitochondria. Furthermore, the mitochondrial complex III activity were inhibited by propamidine.

Hemozoin-free Plasmodium falciparum mitochondria for physiological and drug susceptibility studies.

Isolation of mitochondria of high purity and with intact enzymatic activities from malaria parasites has proven to be a major obstacle in characterizing the parasite mitochondrial physiology. We describe here an improved procedure for the isolation of a mitochondrially enriched preparation from the trophozoite stage of erythrocytic Plasmodium falciparum, combining disruption by N(2) cavitation and differential centrifugation with magnetic removal of hemozoin-associated material. These mitochondrial preparations may be used to assay various mitochondrial enzyme activities, such as succinate and dihydroorotate dehydrogenases, ubiquinol-cytochrome c oxidoreductase, and cytochrome c oxidase. They also exhibit a low level of ATPase activity, which is only marginally inhibited by classical inhibitors. We have used this preparation to determine the susceptibility of mitochondrial activities to drugs and drug candidate compounds in both "wild type" and transgenic parasites.

Effect of acute and chronic administration of methylphenidate on mitochondrial respiratory chain in the brain of young rats.

Methylphenidate is commonly used for the treatment of attention deficit/hyperactivity disorder. There are still few works regarding the effects of methylphenidate on brain energy metabolism. Thus, in the present study we evaluated the effect of chronic administration of methylphenidate on the activities of mitochondrial respiratory chain complexes I and III in the brain of young rats. The effect of acute administration of methylphenidate on mitochondrial respiratory chain complexes I, II, III and IV in the brain of young rats was also investigated. For acute administration, a single injection of methylphenidate was given to rats on postnatal day 25. For chronic administration, methylphenidate injections were given starting at postnatal day 25 once daily for 28 days. Our results showed that complexes I and III were not affected by chronic administration of methylphenidate. Moreover, the acute administration of methylphenidate decreased complex I activity in cerebellum and prefrontal cortex, whereas complexes II, III and IV were not altered.

Ambivalent effects of diazoxide on mitochondrial ROS production at respiratory chain complexes I and III.

BACKGROUND: Reactive oxygen species (ROS) are among the main determinants of cellular damage during ischemia and reperfusion. There is also ample evidence that mitochondrial ROS production is involved in signaling during ischemic and pharmacological preconditioning. In a previous study we analyzed the mitochondrial effects of the efficient preconditioning drug diazoxide and found that it increased the mitochondrial oxidation of the ROS-sensitive fluorescent dye 2',7'-dichlorodihydrofluorescein (H(2)DCF) but had no direct impact on the H(2)O(2) production of submitochondrial particles (SMP) or intact rat heart mitochondria (RHM). METHODS: H(2)O(2) generation of bovine SMP and tightly coupled RHM was monitored under different conditions using the amplex red/horseradish peroxidase assay in response to diazoxide and a number of inhibitors. RESULTS: We show that diazoxide reduces ROS production by mitochondrial complex I under conditions of reverse electron transfer in tightly coupled RHM, but stimulates mitochondrial ROS production at the Q(o) site of complex III under conditions of oxidant-induced reduction; this stimulation is greatly enhanced by uncoupling. These opposing effects can both be explained by inhibition of complex II by diazoxide. 5-Hydroxydecanoate had no effect, and the results were essentially identical in the presence of Na(+) or K(+) excluding a role for putative mitochondrial K(ATP)-channels. GENERAL SIGNIFICANCE: A straightforward rationale is presented to mechanistically explain the ambivalent effects of diazoxide reported in the literature. Depending on the metabolic state and the membrane potential of mitochondria, diazoxide-mediated inhibition of complex II promotes transient generation of signaling ROS at complex III (during preconditioning) or attenuates the production of deleterious ROS at complex I (during ischemia and reperfusion).

Mitochondrial generation of reactive oxygen species is enhanced at the Q(o) site of the complex III in the myocardium of Trypanosoma cruzi-infected mice: beneficial effects of an antioxidant.

In this study, we have characterized the cellular source and mechanism for the enhanced generation of reactive oxygen species (ROS) in the myocardium during Trypanosoma cruzi infection. Cardiac mitochondria of infected mice, as compared to normal controls, exhibited 63.3% and 30.8% increase in ROS-specific fluorescence of dihydroethidium (detects O(2) (*-)) and amplex red (detects H(2)O(2)), respectively. This increase in ROS level in cardiac mitochondria of infected mice was associated with a 59% and 114% increase in the rate of glutamate/malate- (complex I substrates) and succinate- (complex II substrate) supported ROS release, respectively, and up to a 74.9% increase in the rate of electron leakage from the respiratory chain when compared to normal controls. Inhibition studies with normal cardiac mitochondria showed that rotenone induced ROS generation at the Q(Nf)-ubisemiquinone site in complex I. In complex III, myxothiazol induced ROS generation from a site located at the Q(o) center that was different from the Q(i) center of O(2) (*-) generation by antimycin. In cardiac mitochondria of infected mice, the rate of electron leakage at complex I during forward (complex I-to-complex III) and reverse (complex II-to-complex I) electron flow was not enhanced, and complex I was not the main site of increased ROS production in infected myocardium. Instead, defects of complex III proximal to the Q(o) site resulted in enhanced electron leakage and ROS formation in cardiac mitochondria of infected mice. Treatment of infected mice with phenyl-alpha-tert-butyl-nitrone (PBN) improved the respiratory chain function, and, subsequently, decreased the extent of electron leakage and ROS release. In conclusion, we show that impairment of the Q(o) site of complex III resulted in increased electron leakage and O(2) (*-) formation in infected myocardium, and was controlled by PBN.