Reverse Electron Transport Research Articles

Reactive oxygen species production induced by pore opening in cardiac mitochondria: The role of complex II

Succinate-driven reverse electron transport (RET) through complex I is hypothesized to be a major source of reactive oxygen species (ROS) that induces permeability transition pore (PTP) opening and damages the heart during ischemia/reperfusion. Because RET can only generate ROS when mitochondria are fully polarized, this mechanism is self-limiting once PTP opens during reperfusion. In the accompanying article (Korge, P., Calmettes, G., John, S. A., and Weiss, J. N. (2017) J. Biol. Chem. 292, 9882-9895), we showed that ROS production after PTP opening can be sustained when complex III is damaged (simulated by antimycin). Here we show that complex II can also contribute to sustained ROS production in isolated rabbit cardiac mitochondria following inner membrane pore formation induced by either alamethicin or calcium-induced PTP opening. Two conditions are required to maximize malonate-sensitive ROS production by complex II in isolated mitochondria: (a) complex II inhibition by atpenin A5 or complex III inhibition by stigmatellin that results in succinate-dependent reduction of the dicarboxylate-binding site of complex II (site IIf); (b) pore opening in the inner membrane resulting in rapid efflux of succinate/fumarate and other dicarboxylates capable of competitively binding to site IIf The decrease in matrix [dicarboxylate] allows O2 access to reduced site IIf, thereby making electron donation to O2 possible, explaining the rapid increase in ROS production provided that site IIf is reduced. Because ischemia is known to inhibit complexes II and III and increase matrix succinate/fumarate levels, we hypothesize that by allowing dicarboxylate efflux from the matrix, PTP opening during reperfusion may activate sustained ROS production by this mechanism after RET-driven ROS production has ceased.

Mitochondrial complex I deactivation is related to superoxide production in acute hypoxia

Mitochondria use oxygen as the final acceptor of the respiratory chain, but its incomplete reduction can also produce reactive oxygen species (ROS), especially superoxide. Acute hypoxia produces a superoxide burst in different cell types, but the triggering mechanism is still unknown. Herein, we show that complex I is involved in this superoxide burst under acute hypoxia in endothelial cells. We have also studied the possible mechanisms by which complex I could be involved in this burst, discarding reverse electron transport in complex I and the implication of PTEN-induced putative kinase 1 (PINK1). We show that complex I transition from the active to ‘deactive’ form is enhanced by acute hypoxia in endothelial cells and brain tissue, and we suggest that it can trigger ROS production through its Na+/H+ antiporter activity. These results highlight the role of complex I as a key actor in redox signalling in acute hypoxia.

Hypoxia and aerobic metabolism adaptations of human endothelial cells

The goal of our study was to assess the influence of chronic exposure to hypoxia on mitochondrial oxidative metabolism in human umbilical vein endothelial cells (EA.hy926 line) cultured for 6 days at 1% O2 tension. The hypoxia-induced effects were elucidated at the cellular and isolated mitochondria levels. Hypoxia elevated fermentation but did not change mitochondrial biogenesis or the aerobic respiratory capacity of endothelial cells. In endothelial cells, hypoxia caused a general decrease in mitochondrial respiration during carbohydrate, fatty acid, and amino acid oxidation but increased exclusively ketogenic amino acid oxidation. Hypoxia induced an elevation of intracellular and mitochondrial reactive oxygen species (ROS) formation, although cell viability was unchanged and antioxidant systems (superoxide dismutases SOD1 and SOD2, and uncoupling proteins (UCPs)) were not increased. In mitochondria from hypoxic cells, the opposite change was observed at the respiratory chain level, i.e., considerably elevated expression and activity of complex II, and decreased expression and activity of complex I were observed. The elevated activity of complex II resulted in an increase in succinate-sustained mitochondrial ROS formation, mainly through increased reverse electron transport. A hypoxia-induced decrease in UCP2 expression and activity was also observed. It can be concluded that the exposure to chronic hypoxia induces a shift from aerobic toward anaerobic catabolic metabolism. The hypoxia-induced increase in intracellular and mitochondrial ROS formation was not excessive and may be involved in endothelial signaling of hypoxic responses. Our results indicate an important role of succinate, complex II, and reverse electron transport in hypoxia-induced adjustments in endothelial cells.

Anaerobic Formate and Hydrogen Metabolism.

Numerous recent developments in the biochemistry, molecular biology, and physiology of formate and H2 metabolism and of the [NiFe]-hydrogenase (Hyd) cofactor biosynthetic machinery are highlighted. Formate export and import by the aquaporin-like pentameric formate channel FocA is governed by interaction with pyruvate formate-lyase, the enzyme that generates formate. Formate is disproportionated by the reversible formate hydrogenlyase (FHL) complex, which has been isolated, allowing biochemical dissection of evolutionary parallels with complex I of the respiratory chain. A recently identified sulfido-ligand attached to Mo in the active site of formate dehydrogenases led to the proposal of a modified catalytic mechanism. Structural analysis of the homologous, H2-oxidizing Hyd-1 and Hyd-5 identified a novel proximal [4Fe-3S] cluster in the small subunit involved in conferring oxygen tolerance to the enzymes. Synthesis of Salmonella Typhimurium Hyd-5 occurs aerobically, which is novel for an enterobacterial Hyd. The O2-sensitive Hyd-2 enzyme has been shown to be reversible: it presumably acts as a conformational proton pump in the H2-oxidizing mode and is capable of coupling reverse electron transport to drive H2 release. The structural characterization of all the Hyp maturation proteins has given new impulse to studies on the biosynthesis of the Fe(CN)2CO moiety of the [NiFe] cofactor. It is synthesized on a Hyp-scaffold complex, mainly comprising HypC and HypD, before insertion into the apo-large subunit. Finally, clear evidence now exists indicating that Escherichia coli can mature Hyd enzymes differentially, depending on metal ion availability and the prevailing metabolic state. Notably, Hyd-3 of the FHL complex takes precedence over the H2-oxidizing enzymes.

Redox regulation of mitochondrial functional activity by quinones

Quinones are among the rare compounds successfully used as therapeutic agents to correct mitochondrial diseases and as specific regulators of mitochondrial function within cells. The aim of the present study was to elucidate the redox-dependent effects of quinones on mitochondrial function. The functional parameters [respiratory activity, membrane potential, and reactive oxygen species (ROS) generation] of isolated rat liver mitochondria and mitochondria in intact cells were measured in the presence of eight exogenously applied quinones that differ in lipophilicity and one-electron reduction potential. The quinones affected the respiratory parameters of mitochondria, and dissipated the mitochondrial membrane potential as well as influenced (either decreased or enhanced) ROS generation, and restored the electron flow during electron transport chain inhibition. The stimulation of ROS production by juglone and 2,5-di-tert-butyl-1,4-benzoquinone was accompanied by a decrease in the acceptor control and respiration control ratios, dissipation of the mitochondrial membrane potential and induction of the reverse electron flow under succinate oxidation in isolated mitochondria. Menadione and 2,3,5-trimethyl-1,4-benzoquinone, which decreased the mitochondrial ROS generation, did not affect the mitochondrial potential and, vice versa, were capable of restoring electron transport during Complex I inhibition. In intact C6 cells, all the quinones, except for coenzyme Q10, decreased the mitochondrial membrane potential. Juglone, 1,4-benzoquinone, and menadione showed the most pronounced effects. These findings indicate that quinones with the reduction potential values E1/2 in the range from -99 to -260mV were effective redox regulators of mitochondrial electron transport.

39 - Functional Characterization of S1QELs

S1QELs ( S uppressors of site I Q E lectron L eak) are small molecules that specifically suppress production of O2●– and/or H2O2 during reverse electron transport through mitochondrial respiratory complex I (site IQr) without affecting oxidative phosphorylation. They hold great promise as tools to probe mitochondrial superoxide production and as potential pharmaceutical leads. To better characterize these suppressors, a comparative study was conducted using two strucuturally-unrelated S1QELs, S1QEL1 and S1QEL2, and rotenone, the classical electron transport inhibitor at site IQ. We examined their effects on (1) IQr, (2) O2●–/H2O2 production at site IQ during forward electron transport (site IQf), (3) forward electron transport (FET) and (4) reverse electron transport (RET). S1QELs and rotenone suppressed IQr, with relative potency of S1QEL1 > rotenone > S1QEL2. None of the compounds suppressed IQf at any tested concentration. S1QELs were functionally different from rotenone: S1QELs and rotenone suppressed IQr at submicromolar concentrations, but the inhibition of FET and RET by S1QELs was far less potent than that of rotenone. Rotenone also inhibited FET more strongly than RET, where the opposite was true for S1QEL1. S1QEL1 was also found to be different from S1QEL2: S1QEL1 inhibited FET and RET but S1QEL2 did not, which suggests they have different mechanisms.

Understanding and preventing mitochondrial oxidative damage.

Mitochondrial oxidative damage has long been known to contribute to damage in conditions such as ischaemia–reperfusion (IR) injury in heart attack. Over the past years, we have developed a series of mitochondria-targeted compounds designed to ameliorate or determine how this damage occurs. I will outline some of this work, from MitoQ to the mitochondria-targeted S-nitrosating agent, called MitoSNO, that we showed was effective in preventing reactive oxygen species (ROS) formation in IR injury with therapeutic implications. In addition, the protection by this compound suggested that ROS production in IR injury was mainly coming from complex I. This led us to investigate the mechanism of the ROS production and using a metabolomic approach, we found that the ROS production in IR injury came from the accumulation of succinate during ischaemia that then drove mitochondrial ROS production by reverse electron transport at complex I during reperfusion. This surprising mechanism led us to develop further new therapeutic approaches to have an impact on the damage that mitochondrial ROS do in pathology and also to explore how mitochondrial ROS can act as redox signals. I will discuss how these approaches have led to a better understanding of mitochondrial oxidative damage in pathology and also to the development of new therapeutic strategies.

Molecular level distribution of black phosphorus quantum dots on nitrogen-doped graphene nanosheets for superior lithium storage

Abstract Black phosphorus (BP) is a rising star shining in the field of electrochemical energy storage because of its ability to react with up to three lithium to form a Li 3 P compound, giving a theoretical capacity as high as 2596 mA h g −1 . However, inherent embarrassments such as low electronic conductivity and huge volume expansion drastically deteriorate its ultimate electrochemical performances. Although hybridizing BP with a conductive matrix ( e.g. , graphene) is a fascinating concept, its extremely high chemical inertness sets obstacles for constructing reliable interfacial interactions with graphene. Herein we report, for the first time, a facile strategy for the molecular level distribution of BP quantum dots (QDs) on nitrogen-doped graphene ( N -graphene) nanosheets. The small size of the BP QDs translates into abundant active sites, short lithium diffusion pathways and little mechanical fracture, which mean excellent electrochemical kinetics. The molecular level distribution of these QDs on the N -graphene nanosheets effectively prevents their aggregation upon cycling, thereby preserving the whole structure and maintaining good stability. Besides, the N -graphene nanosheets not only buffer the mechanical stress associated with cycling, but also constitute a conductive network to ensure reversible electron transport. The synergistic superiority of the resulting van der Waals heterostructures is well demonstrated by their superior electrochemical performances, delivering a startlingly high reversible capacity of 1271 mA h g −1 at 500 mA g −1 . We believe this strategy may provide a new route to addressing the capacity deficiency which limits the progress of LIBs towards large-size power tools.

Suppressors of Superoxide-H2O2 Production at Site IQ of Mitochondrial Complex I Protect against Stem Cell Hyperplasia and Ischemia-Reperfusion Injury

Using high-throughput screening we identified small molecules that suppress superoxide and/or H2O2 production during reverse electron transport through mitochondrial respiratory complex I (site IQ) without affecting oxidative phosphorylation (suppressors of site IQ electron leak, "S1QELs"). S1QELs diminished endogenous oxidative damage in primary astrocytes cultured at ambient or low oxygen tension, showing that site IQ is a normal contributor to mitochondrial superoxide-H2O2 production in cells. They diminished stem cell hyperplasia in Drosophila intestine invivo and caspase activation in a cardiomyocyte cell model driven by endoplasmic reticulum stress, showing that superoxide-H2O2 production by site IQis involved in cellular stress signaling. They protected against ischemia-reperfusion injury in perfused mouse heart, showing directly that superoxide-H2O2 production by site IQ is a major contributor to this pathology. S1QELs are tools for assessing the contribution of site IQ to cell physiology and pathology and have great potential as therapeutic leads.

Coenzyme Q redox status controls the dynamic configuration of the mitochondrial electron transport chain by reverse electron transport

Coenzyme Q redox status controls the dynamic configuration of the mitochondrial electron transport chain by reverse electron transport

Induction of reverse electron transport in vivo extends lifespan and delays the onset of age-related diseases

Induction of reverse electron transport in vivo extends lifespan and delays the onset of age-related diseases

Succinate metabolism: a new therapeutic target for myocardial reperfusion injury.

Myocardial ischaemia/reperfusion (IR) injury is a major cause of death worldwide and remains a disease for which current clinical therapies are strikingly deficient. While the production of mitochondrial reactive oxygen species (ROS) is a critical driver of tissue damage upon reperfusion, the precise mechanisms underlying ROS production have remained elusive. More recently, it has been demonstrated that a specific metabolic mechanism occurs during ischaemia that underlies elevated ROS at reperfusion, suggesting a unifying model as to why so many different compounds have been found to be cardioprotective against IR injury. This review will discuss the role of the citric acid cycle intermediate succinate in IR pathology focusing on the mechanism by which this metabolite accumulates during ischaemia and how it can drive ROS production at Complex I via reverse electron transport. We will then examine the potential for manipulating succinate accumulation and metabolism during IR injury in order to protect the heart against IR damage and discuss targets for novel therapeutics designed to reduce reperfusion injury in patients.

Effects of excess succinate on cardiomyocyte mitochondrial H2O2 production and cell death

IntroductionDuring myocardial infarction injury, reactive oxygen species (ROS) generation in the first few minutes of reperfusion has been shown to cause significant cellular dysfunction and death of the myocardium. Recent studies suggest ROS generation during reperfusion is due to accumulation of intermediate citric acid cycle metabolites, specifically succinate accumulation. Additionally, previous studies suggest that an increase in fumarate accompanied by an increase in succinate is cardioprotective. In this study, we examined the effect of excess succinate on H2O2 production and cell death. We hypothesized that excess succinate increases H2O2 production through reverse electron transport in mitochondria isolated from Sprague‐Dawley rat hearts and increases cell death in induced pluripotent stem cell‐derived cardiomyocytes (iPSC‐CMs) and that addition of fumarate would abolish this phenotype.MethodsThe left ventricles of Sprague Dawley male rats were collected and minced. Mitochondria were isolated using differential centrifugation. Mitochondria were exposed to 5 mM succinate with or without 1μM rotenone (a complex I inhibitor). H2O2 production was measured using an Amplex Red assay. Human iPSCs were cultured to confluency and differentiated into CMs. Contracting cells were immunostained for cardiac specific markers to confirm >90% purity of CMs. CMs were exposed to 5 mM dimethyl succinate with or without 5 mM dimethyl fumarate for 1 hour followed by 24 hours of reperfusion in basal media. Cell viability was measured using propidium iodide staining.ResultsExcess succinate caused a significant increase in H2O2 production in mitochondria isolated from Sprague Dawley rat hearts. This effect was abolished with addition of rotenone, showing that H2O2 production was occurring through reverse electron transport. Dimethyl succinate also caused significant cell death in iPSC‐derived CMs. Dimethyl fumarate with dimethyl succinate did not abolish increased cell death.ConclusionsWe have confirmed that excess succinate causes a significant increase in H2O2 production through reverse electron transport in mitochondria isolated from Sprague Dawley rat hearts and a significant increase in cell death in iPSC‐derived CMs. Fumarate in addition to excess succinate did not have a cardioprotective effect in iPSC‐CMs. However, this may be a toxic effect rather than a metabolic effect. Further experiments detailing the mechanism by which this occurs may reveal new therapeutic targets to reduce the detriment of ROS production from ischemia/reperfusion injury.Support or Funding InformationNIH/NIGMS Program Project Grant 2P01GM066730‐11Program Director: Zeljko J. Bosnjak “Mechanisms of Anesthetic Cardioprotection”Project II: “MicroRNA in Anesthetic Cardioprotection”

Mitochondrial ROS Produced via Reverse Electron Transport Extend Animal Lifespan

SummaryIncreased production of reactive oxygen species (ROS) has long been considered a cause of aging. However, recent studies have implicated ROS as essential secondary messengers. Here we show that the site of ROS production significantly contributes to their apparent dual nature. We report that ROS increase with age as mitochondrial function deteriorates. However, we also demonstrate that increasing ROS production specifically through respiratory complex I reverse electron transport extends Drosophila lifespan. Reverse electron transport rescued pathogenesis induced by severe oxidative stress, highlighting the importance of the site of ROS production in signaling. Furthermore, preventing ubiquinone reduction, through knockdown of PINK1, shortens lifespan and accelerates aging; phenotypes that are rescued by increasing reverse electron transport. These results illustrate that the source of a ROS signal is vital in determining its effects on cellular physiology and establish that manipulation of ubiquinone redox state is a valid strategy to delay aging.

The CoQH2/CoQ Ratio Serves as a Sensor of Respiratory Chain Efficiency.

Electrons feed into the mitochondrial electron transport chain (mETC) from NAD- or FAD-dependent enzymes. A shift from glucose to fatty acids increases electron flux through FAD, which can saturate the oxidation capacity of the dedicated coenzyme Q (CoQ) pool and result in the generation of reactive oxygen species. To prevent this, the mETC superstructure can be reconfigured through the degradation of respiratory complex I, liberating associated complex III to increase electron flux via FAD at the expense of NAD. Here, we demonstrate that this adaptation is driven by the ratio of reduced to oxidized CoQ. Saturation of CoQ oxidation capacity induces reverse electron transport from reduced CoQ to complex I, and the resulting local generation of superoxide oxidizes specific complex I proteins, triggering their degradation and the disintegration of the complex. Thus, CoQ redox status acts as a metabolic sensor that fine-tunes mETC configuration in order to match the prevailing substrate profile.

The bright side of reactive oxygen species: lifespan extension without cellular demise.

Oxidative stress and the generation of reactive oxygen species (ROS) can lead to mitochondrial dysfunction, DNA damage, protein misfolding, programmed cell death with apoptosis and autophagy, and the promotion of aging –dependent processes. Mitochondria control the processing of redox energy that yields adenosine triphosphate (ATP) through the oxidation of glucose, pyruvate, and nicotinamide adenine dinucleotide. Ultimately, the generation of ROS occurs with the aerobic production of ATP. Although reduced levels of ROS may lead to tolerance against metabolic, mechanical, and oxidative stressors and the generation of brief periods of ROS during ischemia-reperfusion models may limit cellular injury, under most circumstances ROS and mitochondrial dysfunction can lead to apoptotic caspase activation and autophagy induction that can result in cellular demise. Yet, new work suggests that ROS generation may have a positive impact through respiratory complex I reverse electron transport that can extend lifespan. Such mechanisms may bring new insight into clinically relevant disorders that are linked to cellular senescence and aging of the body’s system. Further investigation of the potential “bright side” of ROS and mitochondrial respiration is necessary to target specific pathways, such as the mechanistic target of rapamycin, nicotinamidases, sirtuins, mRNA decoupling and protein expression, and Wnt signaling, that can impact oxidative stress-ROS mechanisms to extend lifespan and eliminate disease onset.

Mechanism and protective properties of S-nitrosation by nitric oxide in cardiac mitochondria

Oxidative damage from elevated production of reactive oxygen species (ROS) contributes to ischemia-reperfusion injury (IRI) in many pathologies, such as myocardial infarction, stroke, and organ transplantation. We recently determined a critical mechanism for protection from IRI by nitric oxide (NO)-dependent S-nitrosation using a mitochondria-selective S-nitrosating agent, MitoSNO. MitoSNO contains an S-nitrosating moiety derived from the NO-donor SNAP, attached to a lipophilic triphenylphosphonium cation that leads to its rapid and extensive uptake within mitochondria in vivo. Protection by MitoSNO is afforded by inhibition of complex I through selective S-nitrosation of Cys39 on the ND3 subunit, which becomes susceptible to modification only after ischaemia. The observed posttranslational alteration of complex I slows the reactivation of mitochondria during the crucial first minutes of the reperfusion of ischemic tissue, thereby decreasing ROS production, oxidative damage and tissue necrosis. Furthermore, we could show that the reperfusion-induced ROS production is not an unspecific event of complex I reactivation but is highly regulated by a dramatic increase in succinate during ischaemia. ROS are subsequently produced via reverse electron transport and inhibition of the succinate rise during ischaemia proved to be a further promising target to protect against IRI injury. Taken together, reversible S-nitrosation of complex I or the prevention of ischaemic succinate accumulation results in significant protection from acute and long-term effects of IRI and provide evidence for compounds selectively targeting the mitochondria, such as MitoSNO, to be a promising therapeutic strategy for translation to use in humans.

Abstract 404: High-throughput Screening Reveals the Mitochondrial Complex I Inhibitor Nornicotine is Cardioprotective in Ischemia-reperfusion Injury When Delivered at Reperfusion

Background: To date, there are no FDA-approved therapies for the reduction of infarct size in acute myocardial infarction. Previously, we developed a cell-based phenotypic assay of ischemia-reperfusion (IR) injury, which was used to identify novel cytoprotective agents delivered prior to ischemia. Herein, we sought to identify cytoprotective agents in a more clinically relevant model: drug delivery at reperfusion, and to investigate possible underlying mechanisms of protection. Methods: Primary adult mouse cardiomyocytes were subjected to simulated IR injury using a modified Seahorse XF24 apparatus with drug addition at the onset of reperfusion. Cell death was estimated using LDH release. Drugs which protected cardiomyocytes in vitro were tested in a Langendorff model of IR injury, measuring functional recovery and infarct size. In separate experiments, metabolites extracted from perfused hearts were resolved by HPLC. Results: Nornicotine was identified as a cardioprotective agent in the screen. In perfused hearts, 10 nM nornicotine injected at the onset of reperfusion improved functional recovery and decreased in infarct size (13.1% ± 2.4 vs 49.2% ± 2.5 in non-treated hearts, p<0.05, n=16-20). Nornicotine also exhibited profound inhibitory effects on mitochondrial complex I activity. Succinate is known to accumulate in ischemia, and its rapid consumption during early reperfusion exacerbates reperfusion injury via ROS generation from electron backflow through complex I [PMID: 25383517]. In non-treated hearts, we confirmed that high post ischemic levels of succinate rapidly declined during the first 2 min of reperfusion. In contrast, nornicotine slowed post-ischemic succinate consumption, suggesting that electron backflow through complex I is the major pathway driving succinate consumption. Conclusions: Herein, we demonstrated that nornicotine was cardioprotective when delivered at early reperfusion in vitro and ex vivo. The mechanism of cardioprotection may be due to inhibition of rapid succinate consumption during early reperfusion via reverse electron flow back through complex I.

The aerobic respiratory chain of the acidophilic archaeon Ferroplasma acidiphilum: A membrane-bound complex oxidizing ferrous iron

The extremely acidophilic archaeon Ferroplasma acidiphilum is found in iron-rich biomining environments and is an important micro-organism in naturally occurring microbial communities in acid mine drainage. F. acidiphilum is an iron oxidizer that belongs to the order Thermoplasmatales (Euryarchaeota), which harbors the most extremely acidophilic micro-organisms known so far. At present, little is known about the nature or the structural and functional organization of the proteins in F. acidiphilum that impact the iron biogeochemical cycle. We combine here biochemical and biophysical techniques such as enzyme purification, activity measurements, proteomics and spectroscopy to characterize the iron oxidation pathway(s) in F. acidiphilum. We isolated two respiratory membrane protein complexes: a 850kDa complex containing an aa3-type cytochrome oxidase and a blue copper protein, which directly oxidizes ferrous iron and reduces molecular oxygen, and a 150kDa cytochrome ba complex likely composed of a di-heme cytochrome and a Rieske protein. We tentatively propose that both of these complexes are involved in iron oxidation respiratory chains, functioning in the so-called uphill and downhill electron flow pathways, consistent with autotrophic life. The cytochrome ba complex could possibly play a role in regenerating reducing equivalents by a reverse (‘uphill’) electron flow. This study constitutes the first detailed biochemical investigation of the metalloproteins that are potentially directly involved in iron-mediated energy conservation in a member of the acidophilic archaea of the genus Ferroplasma.

The calcium-ROS-pH triangle and mitochondrial permeability transition: challenges to mimic cardiac ischemia-reperfusion.

The calcium-ROS-pH triangle and mitochondrial permeability transition: challenges to mimic cardiac ischemia-reperfusion.