The fungicide cymoxanil impairs respiration in Saccharomyces cerevisiae via cytochrome c oxidase inhibition.

Cymoxanil (CYM) is a widely used synthetic acetamide fungicide, but its biochemical mode of action remains elusive. Since CYM inhibits cell growth, biomass production, and respiration in Saccharomyces cerevisiae, we used this model to characterize the effect of CYM on mitochondria. We found it inhibits oxygen consumption in both whole cells and isolated mitochondria, specifically inhibiting cytochrome c oxidase (CcO) activity during oxidative phosphorylation. Based on molecular docking, we propose that CYM blocks the interaction of cytochrome c with CcO, hampering electron transfer and inhibiting CcO catalytic activity. Although other targets cannot be excluded, our data offer valuable insights into the mode of action of CYM that will be instrumental in driving informed management of the use of this fungicide.

Identification of 2-Aryl-Quinolone Inhibitors of Cytochrome bd and Chemical Validation of Combination Strategies for Respiratory Inhibitors against Mycobacterium tuberculosis.

Mycobacterium tuberculosis cytochrome bd quinol oxidase (cyt bd), the alternative terminal oxidase of the respiratory chain, has been identified as playing a key role during chronic infection and presents a putative target for the development of novel antitubercular agents. Here, we report confirmation of successful heterologous expression of M. tuberculosis cytochrome bd. The heterologous M. tuberculosis cytochrome bd expression system was used to identify a chemical series of inhibitors based on the 2-aryl-quinolone pharmacophore. Cytochrome bd inhibitors displayed modest efficacy in M. tuberculosis growth suppression assays together with a bacteriostatic phenotype in time-kill curve assays. Significantly, however, inhibitor combinations containing our front-runner cyt bd inhibitor CK-2-63 with either cyt bcc-aa3 inhibitors (e.g., Q203) and/or adenosine triphosphate (ATP) synthase inhibitors (e.g., bedaquiline) displayed enhanced efficacy with respect to the reduction of mycobacterium oxygen consumption, growth suppression, and in vitro sterilization kinetics. In vivo combinations of Q203 and CK-2-63 resulted in a modest lowering of lung burden compared to treatment with Q203 alone. The reduced efficacy in the in vivo experiments compared to in vitro experiments was shown to be a result of high plasma protein binding and a low unbound drug exposure at the target site. While further development is required to improve the tractability of cyt bd inhibitors for clinical evaluation, these data support the approach of using small-molecule inhibitors to target multiple components of the branched respiratory chain of M. tuberculosis as a combination strategy to improve therapeutic and pharmacokinetic/pharmacodynamic (PK/PD) indices related to efficacy.

Structural basis of mammalian complex IV inhibition by steroids.

The mitochondrial electron transport chain maintains the proton motive force that powers adenosine triphosphate (ATP) synthesis. The energy for this process comes from oxidation of reduced nicotinamide adenine dinucleotide (NADH) and succinate, with the electrons from this oxidation passed via intermediate carriers to oxygen. Complex IV (CIV), the terminal oxidase, transfers electrons from the intermediate electron carrier cytochrome c to oxygen, contributing to the proton motive force in the process. Within CIV, protons move through the K and D pathways during turnover. The former is responsible for transferring two protons to the enzyme's catalytic site upon its reduction, where they eventually combine with oxygen and electrons to form water. CIV is the main site for respiratory regulation, and although previous studies showed that steroid binding can regulate CIV activity, little is known about how this regulation occurs. Here, we characterize the interaction between CIV and steroids using a combination of kinetic experiments, structure determination, and molecular simulations. We show that molecules with a sterol moiety, such as glyco-diosgenin and cholesteryl hemisuccinate, reversibly inhibit CIV. Flash photolysis experiments probing the rapid equilibration of electrons within CIV demonstrate that binding of these molecules inhibits proton uptake through the K pathway. Single particle cryogenic electron microscopy (cryo-EM) of CIV with glyco-diosgenin reveals a previously undescribed steroid binding site adjacent to the K pathway, and molecular simulations suggest that the steroid binding modulates the conformational dynamics of key residues and proton transfer kinetics within this pathway. The binding pose of the sterol group sheds light on possible structural gating mechanisms in the CIV catalytic cycle.

Metformin, phenformin, and galegine inhibit complex IV activity and reduce glycerol-derived gluconeogenesis.

SignificanceMetformin is the most commonly prescribed drug for the treatment of type 2 diabetes mellitus, yet the mechanism by which it lowers plasma glucose concentrations has remained elusive. Most studies to date have attributed metformin's glucose-lowering effects to inhibition of complex I activity. Contrary to this hypothesis, we show that inhibition of complex I activity in vitro and in vivo does not reduce plasma glucose concentrations or inhibit hepatic gluconeogenesis. We go on to show that metformin, and the related guanides/biguanides, phenformin and galegine, inhibit complex IV activity at clinically relevant concentrations, which, in turn, results in inhibition of glycerol-3-phosphate dehydrogenase activity, increased cytosolic redox, and selective inhibition of glycerol-derived hepatic gluconeogenesis both in vitro and in vivo.

Complexities of complex II: Sulfide metabolism in vivo.

High levels of H2S produced by gut microbiota can block oxygen utilization by inhibiting mitochondrial complex IV. Kumar et al. have shown how cells respond to this inhibition by using the mitochondrial sulfide oxidation pathway and reverse electron transport. The reverse activity of mitochondrial complex II (succinate-quinone oxidoreductase, i.e., fumarate reduction) generates oxidized coenzyme Q, which is then reduced by the mitochondrial sulfide quinone oxidoreductase to oxidize H2S. This newly identified redox circuitry points to the importance of complex II reversal in mitochondria during periods of hypoxia and cellular stress.

A redox cycle with complex II prioritizes sulfide quinone oxidoreductase-dependent H2S oxidation.

The dual roles of H2S as an endogenously synthesized respiratory substrate and as a toxin raise questions as to how it is cleared when the electron transport chain is inhibited. Sulfide quinone oxidoreductase (SQOR) catalyzes the first step in the mitochondrial H2S oxidation pathway, using CoQ as an electron acceptor, and connects to the electron transport chain at the level of complex III. We have discovered that at high H2S concentrations, which are known to inhibit complex IV, a new redox cycle is established between SQOR and complex II, operating in reverse. Under these conditions, the purine nucleotide cycle and the malate aspartate shuttle furnish fumarate, which supports complex II reversal and leads to succinate accumulation. Complex II knockdown in colonocytes decreases the efficiency of H2S clearance while targeted knockout of complex II in intestinal epithelial cells significantly decreases the levels of thiosulfate, a biomarker of H2S oxidation, to approximately one-third of the values seen in serum and urine samples from control mice. These data establish the physiological relevance of this newly discovered redox circuitry between SQOR and complex II for prioritizing H2S oxidation and reveal the quantitatively significant contribution of intestinal epithelial cells to systemic H2S metabolism.

Antidotal effect of dihydroxyacetone against phosphine poisoning in mice.

Phosphine (PH3 ) is widely used as an insecticide and rodenticide. On the contrary, many cases of PH3 poisoning have been reported worldwide. Unfortunately, there is no specific antidote against PH3 toxicity. Disruption of mitochondrial function and energy metabolism is a well-known mechanism of PH3 cytotoxicity. Dihydroxyacetone (DHA) is an adenosine triphosphate supplying agent which significantly improves mitochondrial function. The current study was designed to evaluate DHA's effect on inhalational PH3 poisoning in an animal model. DHA was injected into BALB/c mice before and/or after the start of the PH3 inhalation. The cytochrome c oxidase activity was assessed in the animals' brain, heart, and liver exposed to PH3 (for 15, 30, and 60 min, with and without the antidote). The LC50 of PH3 was calculated to be 18.02 (15.42-20.55) ppm over 2 h of exposure. Pretreatment of DHA (1 or 2 g/kg) increased the LC50 of PH3 by about 1.6- or 3-fold, respectively. Posttreatment with DHA (2 g/kg) increased the LC50 of PH3 by about 1.4-fold. PH3 inhibited the activity of cytochrome c oxidase in the assessed organs. It was found that DHA treatment restored mitochondrial cytochrome c oxidase activity. These findings suggested that DHA could be an effective antidote for PH3 poisoning.

Mitochondrial toxins potentiate hydroxyl radical production in rat striatum during carbon monoxide poisoning.

Hydroxyl radical (•OH) production in the rat striatum during carbon monoxide (CO) poisoning, which inhibits complex IV, was enhanced synergistically by malonate, a mitochondrial complex II inhibitor, but not N-methyl-4-phenylpyridinium or NaCN, complex I and IV inhibitors, respectively. No such enhancement appeared in the case of NaCN combined with malonate. Intrastriatal dopamine, which is involved in •OH production by malonate, did not synergistically enhance CO-induced •OH production. Diphenyleneiodonium, a nonselective NADPH oxidase inhibitor, partly suppressed the potentiation of CO-induced •OH production by malonate. Impairment of mitochondrial functions might potentiate oxidative stress and intensify CO toxicity in the brain.

A membrane arm of mitochondrial complex I sufficient to promote respirasome formation.

The assembly pathways of mitochondrial respirasome (supercomplex I+III2+IV) are not fully understood. Here, we show that an early sub-complex I assembly, rather than holo-complex I, is sufficient to initiate mitochondrial respirasome assembly. We find that a distal part of the membrane arm of complex I (PD-a module) is a scaffold for the incorporation of complexes III and IV to form a respirasome subcomplex. Depletion of PD-a, rather than other complex I modules, decreases the steady-state levels of complexes III and IV. Both HEK293T cells lacking TIMMDC1 and patient-derived cells with disease-causing mutations in TIMMDC1 showed accumulation of this respirasome subcomplex. This suggests that TIMMDC1, previously known as a complex-I assembly factor, may function as a respirasome assembly factor. Collectively, we provide a detailed, cooperative assembly model in which most complex-I subunits are added to the respirasome subcomplex in the lateral stages of respirasome assembly.

SILAC-based complexome profiling dissects the structural organization of the human respiratory supercomplexes in SCAFIKO cells.

The study of the mitochondrial respiratory chain (MRC) function in relation with its structural organization is of great interest due to the central role of this system in eukaryotic cell metabolism. The complexome profiling technique has provided invaluable information for our understanding of the composition and assembly of the individual MRC complexes, and also of their association into larger supercomplexes (SCs) and respirasomes. The formation of the SCs has been highly debated, and their assembly and regulation mechanisms are still unclear. Previous studies demonstrated a prominent role for COX7A2L (SCAFI) as a structural protein bridging the association of individual MRC complexes III and IV in the minor SC III2 + IV, although its relevance for respirasome formation and function remains controversial. In this work, we have used SILAC-based complexome profiling to dissect the structural organization of the human MRC in HEK293T cells depleted of SCAFI (SCAFIKO) by CRISPR-Cas9 genome editing. SCAFI ablation led to a preferential loss of SC III2 + IV and of a minor subset of respirasomes without affecting OXPHOS function. Our data suggest that the loss of SCAFI-dependent respirasomes in SCAFIKO cells is mainly due to alterations on early stages of CI assembly, without impacting the biogenesis of complexes III and IV. Contrary to the idea of SCAFI being the main player in respirasome formation, SILAC-complexome profiling showed that, in wild-type cells, the majority of respirasomes (ca. 70%) contained COX7A2 and that these species were present at roughly the same levels when SCAFI was knocked-out. We thus demonstrate the co-existence of structurally distinct respirasomes defined by the preferential binding of complex IV via COX7A2, rather than SCAFI, in human cultured cells.

Mechanism of Inhibition of Cytochrome c Oxidase by Triton X-100.

It is known that Triton X-100 (TX) reversibly inhibits activity of cytochrome c oxidase (CcO). The mechanism of inhibition is analyzed in this work. The action of TX is not directed to the reaction of CcO with cytochrome c, does not cause transition of the enzyme to the "slow" form, and is not associated with monomerization of the enzyme complex. TX completely suppresses oxygen reduction by CcO, but inhibition is prevented and partially reversed by dodecyl-ß-D-maltoside (DDM), a detergent used to maintain CcO in solution. A 1/1 stoichiometry competition is shown between DDM and TX for binding to CcO, with Ki = 0.3 mM and affinity of DDM for the enzyme of 1.2 mM. TX interaction with the oxidized enzyme induces spectral response with maximum at 421 nm and [TX]1/2 = 0.28 mM, presumably associated with heme a3. When CcO interacts with excess of H2O2 TX affects equilibrium of the oxygen intermediates of the catalytic center accelerating the FI-607 → FII-580 transition, inhibits generation of O2·- by the enzyme, and, to a lesser extent, suppresses the catalase partial activity. The observed effects can be explained by inhibition of the conversion of the intermediate FII-580 to the free oxidized state during the catalytic cycle. TX suppresses intraprotein electron transfer between hemes a and a3 during enzyme turnover. Partial peroxidase activity of CcO remains relatively resistant to TX under conditions that block oxidase reaction effectively. These features indicate an impairment of the K proton channel conductivity. We suggest that TX interacts with CcO at the Bile Acid Binding Site (BABS) that is located on the subunit I at the K-channel mouth and contacts with amphipathic regulators of CcO [Buhrow et al. (2013) Biochemistry, 52, 6995-7006]. Apparently, TX mimics the physiological ligand of BABS, whereas the DDM molecule mimics an endogenous phospholipid bound at the edge of BABS that controls effective affinity for the ligand.

Role of mitochondrial complex III/IV in the activation of transcription factor Rst2 in Schizosaccharomyces pombe.

Mitochondria play essential roles in eukaryotic cells for glucose metabolism to produce ATP. In Schizosaccharomyces pombe, transcription factor Rst2 can be activated upon glucose deprivation. However, the link between Rst2 and mitochondrial function remains elusive. Here, we monitored Rst2 transcriptional activity in living cells using a Renilla luciferase reporter system, and found that inhibition of mitochondrial complex III/IV caused cells to produce reactive oxygen species (ROS) and nitric oxide (NO), which in turn activated Rst2. Furthermore, Rst2-GFP was observed to translocate from cytoplasm to nucleus upon mitochondrial complex III/IV inhibitors treatment, and deletion of genes associated with complex III/IV resulted in delayed process of Rst2-GFP nuclear exportation under glucose-rich condition. In particular, we found that Rst2 was phosphorylated following the treatment of complex III/IV inhibitors or SNAP. Altogether, our findings suggest that mitochondrial complex III/IV participates in the activation of Rst2 through ROS and NO generation in Schizosaccharomyces pombe.

Cytochrome c oxidase-modulatory near-infrared light penetration into the human brain: Implications for the noninvasive treatment of ischemia/reperfusion injury.

Near-infrared light (IRL) has been evaluated as a therapeutic for a variety of pathological conditions, including ischemia/reperfusion injury of the brain, which can be caused by an ischemic stroke or cardiac arrest. Strategies have focused on modulating the activity of mitochondrial electron transport chain (ETC) enzyme cytochrome c oxidase (COX), which has copper centers that broadly absorb IRL between 700 and 1,000 nm. We have recently identified specific COX-inhibitory IRL wavelengths that are profoundly neuroprotective in rodent models of brain ischemia/reperfusion through the following mechanism: COX inhibition by IRL limits mitochondrial membrane potential hyperpolarization during reperfusion, which otherwise causes reactive oxygen species (ROS) production and cell death. Prior to clinical application of IRL on humans, IRL penetration must be tested, which may be wavelength dependent. In the present study, four fresh (unfixed) cadavers and isolated cadaver tissues were used to examine the transmission of infrared light through human biological tissues. We conclude that the transmission of 750 and 940 nm IRL through 4 cm of cadaver head supports the viability of IRL to treat human brain ischemia/reperfusion injury and is similar for skin with different skin pigmentation. We discuss experimental difficulties of working with fresh cadavers and strategies to overcome them as a guide for future studies.

Effect of acute mitochondrial dysfunction on hyperexcitable network activity in rat hippocampus in vitro.

Metabolic stress imposed by epileptic seizures can result in mitochondrial dysfunction, believed to act as positive feedback on epileptogenesis and seizure susceptibility. As the mechanism behind this positive feedback is unclear, the aim of the present study was to investigate the causal link between acute mitochondrial dysfunction and increased seizure susceptibility in hyperexcitable hippocampal networks. Following the induction of spontaneous interictal-like discharges, acute selective pharmacological blockade of either of the mitochondrial respiratory complexes (MRC) I-IV induced seizure-like events (SLE) in 78-100% of experiments. A similar result was obtained by uncoupling the oxidative phosphorylation (OXPHOS) but not by selective blockade of MRCV (ATP synthase) which did not induce SLE. The reactive oxygen species (ROS) scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (tempol, 2 mM) significantly reduced the proconvulsant effect of blocking MRCI but did not reduce the proconvulsant effect of OXPHOS uncoupling. These findings indicate that acute mitochondrial dysfunction can lead to a convulsive state within a short timeframe, and that increased ROS production makes substantial contribution to such induction in addition to other mitochondrial related factors, which appears to be independent of changes in ROS and ATP production.

Interaction of Cytochrome C Oxidase with Steroid Hormones.

Estradiol, testosterone and other steroid hormones inhibit cytochrome c oxidase (CcO) purified from bovine heart. The inhibition is strongly dependent on concentration of dodecyl-maltoside (DM) in the assay. The plots of Ki vs [DM] are linear for both estradiol and testosterone which may indicate an 1:1 stoichiometry competition between the hormones and the detergent. Binding of estradiol, but not of testosterone, brings about spectral shift of the oxidized CcO consistent with an effect on heme a33+. We presume that the hormones bind to CcO at the bile acid binding site described by Ferguson-Miller and collaborators. Estradiol is shown to inhibit intraprotein electron transfer between hemes a and a3. Notably, neither estradiol nor testosterone suppresses the peroxidase activity of CcO. Such a specific mode of action indicates that inhibition of CcO activity by the hormones is associated with impairing proton transfer via the K-proton channel.

Donepezil Prevents Inhibition of Cerebral Energetic Metabolism Without Altering Behavioral Parameters in Animal Model of Obesity.

Obesity is characterized by chronic inflammation of low grade. The cholinergic anti-inflammatory pathway favors the reduction of the inflammatory response. In this work the effect of stimulation of the cholinergic anti-inflammatory pathway on SHIRPA behavioral test and mitochondrial respiratory chain activity in obese mice was evaluated. The animals were paired in four groups: saline + control diet; donepezil + control diet; saline + high-fat diet and donepezil + high-fat diet. 5 mg/kg/day orally of donepezil or saline were given 7 days before the beginning of the diet until completing 11 weeks of the experiment. Food intake and body weight were measured. At the end of the experiment the animals were submitted to the SHIRPA behavioral test, soon after they were killed by decapitation, the open abdominal cavity and the mesenteric fat were removed. The hypothalamus, hippocampus, prefrontal cortex, and striatum were removed for evaluation of the mitochondrial respiratory chain. It can be observed that donepezil prevented weight gain and food consumption, as well as a tendency to prevent the accumulation of mesenteric fat in obese animals. There was no behavioral change in obese animals, nor did the influence of donepezil on these parameters. On the other hand, donepezil did not prevent inhibition of complex I activity, prevented the inhibition of complex II, and showed a tendency to prevent IV complex activity inhibited in obesity. With these results it can be concluded that the activation of the cholinergic anti-inflammatory pathway is promising for the alterations found in obesity.

miR-204/COX5A axis contributes to invasion and chemotherapy resistance in estrogen receptor-positive breast cancers.

Breast cancer is the most common cancer among women worldwide, with 70% being estrogen receptor-positive (ER+). Although ER-targeted treatment is effective in treating ER + breast cancer, chemoresistance and metastasis still prevail. Outcome-predictable biomarkers can help improve patient prognosis. Through the analysis of the Array Express database, The Cancer Genome Atlas-Breast Cancer datasets, and breast tumor tissue array results, we found that cytochrome c oxidase subunit 5a (COX5A) was related to poor prognosis of ER + breast cancer. Further studies revealed that COX5A was positively associated with metastasis and chemoresistance in ER + breast cancer. In vitro experiments showed that knockdown of COX5A was accompanied by a decrease in ERα expression, cell cycle arrest, and epithelial-mesenchymal transition blockade, resulting in an inhibition of proliferation and invasion. Knockdown of COX5A enhanced the chemosensitivity of breast cancer cells by decreasing adenosine triphosphate and increasing reactive oxygen species levels. We report that miR-204 can target and inhibit the expression of COX5A, thus, reversing the functions of COX5A in ER + breast cancer cells. We found that COX5A may serve as a prognostic biomarker in ER + breast cancer.

Itaconic acid impairs the mitochondrial function by the inhibition of complexes II and IV and induction of the permeability transition pore opening in rat liver mitochondria.

Itaconic acid (methylene-succinic acid, ItA) is an unsaturated dicarboxylic acid that is secreted by mammalian macrophages in response to a pro-inflammatory stimulus and shows an anti-inflammatory/antibacterial effect. Being a mitochondrial metabolite, it exhibits an inhibitory activity on succinate dehydrogenase and subsequently induces mitochondrial dysfunction. The present study has shown that ItA dose-dependently inhibited ADP- and DNP-stimulated (uncoupled) respiration of rat liver mitochondria energized with succinate. This effect of ItA could be related to the suppression of the activity of complex II and the combined activity of complexes II + III of the respiratory chain. At the same time, ItA had no effect on the activity of the dicarboxylate carrier, which catalyzes the transport of succinate across the inner mitochondrial membrane. It was found that 4 mM ItA diminished the rates of ADP- and DNP-stimulated mitochondrial respiration supported by the substrates of complex I glutamate and malate. A study of the effect of ItA on the activity of complexes of the respiratory chain showed that it decreases the activity of complex IV. It was observed that 4 mM ItA inhibited the rate of H2O2 production by mitochondria. At the same time, ItA promoted the opening of the cyclosporin A-sensitive Ca2+-dependent permeability transition pore. The latter was revealed as the decrease in the calcium retention capacity of mitochondria and the stimulation of release of cytochrome c from the organelles. ItA by itself promoted the cytochrome c release from mitochondria. Possible mechanisms of the effect of ItA on mitochondrial function are discussed.

Antituberculosis Activity of the Antimalaria Cytochrome bcc Oxidase Inhibitor SCR0911.

The ability to respire and generate adenosine triphosphate (ATP) is essential for the physiology, persistence, and pathogenicity of Mycobacterium tuberculosis, which causes tuberculosis. By employing a lead repurposing strategy, the malarial cytochrome bc1 inhibitor SCR0911 was tested against mycobacteria. Docking studies were carried out to reveal potential binding and to understand the binding interactions with the target, cytochrome bcc. Whole-cell-based and in vitro assays demonstrated the potency of SCR0911 by inhibiting cell growth and ATP synthesis in both the fast- and slow-growing M. smegmatis and M. bovis bacillus Calmette-Guérin, respectively. The variety of biochemical assays and the use of a cytochrome bcc deficient mutant strain validated the cytochrome bcc oxidase as the direct target of the drug. The data demonstrate the broad-spectrum activity of SCR0911 and open the door for structure-activity relationship studies to improve the potency of new mycobacteria specific SCR0911 analogues.

A Comparison of Potential Azide Antidotes in a Mouse Model.

Three cobalt-containing macrocyclic compounds previously shown to antagonize cyanide toxicity have been comparatively evaluated for the amelioration of sublethal azide toxicity in juvenile (7-8 weeks) Swiss-Webster mice. The lowest effective doses were determined for hydroxocobalamin, a cobalt porphyrin, and a cobalt-Schiff base macrocycle by giving the antidotes 5 min prior to the toxicant, 27 mg (415 µmol) /kg sodium azide. Both male and female mice were evaluated for their response to the toxicant as well as the antidotes, and no significant differences were noted once weight differences were taken into account. Two of the three compounds significantly decreased the recovery time of azide-intoxicated mice at 10 min after the administration of sodium azide, as determined by a behavioral test (pole climbing). Additionally, azide was determined to cause a several degree drop (∼3 °C) in measured tail temperature, and warming the mice led to a more rapid recovery. The mice were also shown to recover more rapidly when given sodium nitrite, 24 mg (350 µmol)/kg, 5 min after the toxicant; this treatment also suppressed the azide-induced tail temperature decrease. Electron paramagnetic resonance (EPR) measurements of mouse blood treated with sodium azide demonstrated the presence of nitrosylhemoglobin at levels of 10-20 µM which persisted for ∼300 min. The presence of the methemoglobin azide adduct was also detected by EPR at a maximum level of ∼300 µM, but these signals disappeared around 200 min after the administration of azide. The treatment of mice with 15N sodium azide proved that the nitrosylhemoglobin was a product of the administered azide by the appearance of a two-line hyperfine (due to the 15N) in the EPR spectrum of mouse blood.