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1.  Superoxide Is Produced by the Reduced Flavin in Mitochondrial Complex I 
The Journal of Biological Chemistry  2011;286(20):18056-18065.
NADH:ubiquinone oxidoreductase (complex I) is a major source of reactive oxygen species in mitochondria and a contributor to cellular oxidative stress. In isolated complex I the reduced flavin is known to react with molecular oxygen to form predominantly superoxide, but studies using intact mitochondria contend that superoxide may result from a semiquinone species that responds to the proton-motive force (Δp) also. Here, we use bovine heart submitochondrial particles to show that a single mechanism describes superoxide production by complex I under all conditions (during both NADH oxidation and reverse electron transfer). NADH-induced superoxide production is inhibited by complex I flavin-site inhibitors but not by inhibitors of ubiquinone reduction, and it is independent of Δp. Reverse electron transfer (RET) through complex I in submitochondrial particles, driven by succinate oxidation and the Δp created by ATP hydrolysis, reduces the flavin, leading to NAD+ and O2 reduction. RET-induced superoxide production is inhibited by both flavin-site and ubiquinone-reduction inhibitors. The potential dependence of NADH-induced superoxide production (set by the NAD+ potential) matches that of RET-induced superoxide production (set by the succinate potential and Δp), and they both match the potential dependence of the flavin. Therefore, both NADH- and RET-induced superoxide are produced by the flavin, according to the same molecular mechanism. The unified mechanism describes how reactive oxygen species production by complex I responds to changes in cellular conditions. It establishes a route to understanding causative connections between the enzyme and its pathological effects and to developing rational strategies for addressing them.
PMCID: PMC3093879  PMID: 21393237
Bioenergetics; Enzyme Mechanisms; Flavoproteins; Membrane Energetics; Mitochondria; Oxidative Stress; Proton Pumps; Reactive Oxygen Species (ROS); Respiratory Chain; NADH:ubiquinone Oxidoreductase
2.  Reactive Oxygen Species Production by Forward and Reverse Electron Fluxes in the Mitochondrial Respiratory Chain 
PLoS Computational Biology  2011;7(3):e1001115.
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.
Author Summary
Respiration at the level of mitochondria is considered as delivery of electrons and protons from NADH or succinate to oxygen through a set of transporters constituting the respiratory chain (RC). Mitochondrial respiration, dealing with transfer of unpaired electrons, may produce reactive oxygen species (ROS) such as O2− and subsequently H2O2 as side products. ROS are chemically very active and can cause oxidative damage to cellular components. The production of ROS, normally low, can increase under stress to the levels incompatible with cell survival; thus, understanding the ways of ROS production in the RC represents a vital task in research. We used mathematical modeling to analyze experiments with isolated brain mitochondria aimed to study relations between electron transport and ROS production. Elsewhere we reported that mitochondrial complex III can operate in two distinct steady states at the same microenvironmental conditions, producing either low or high levels of ROS. Here, this property of bistability was confirmed for the whole RC. The associations between measured ROS production and computed individual free radical levels in complexes I and III were established. The discovered phenomenon of bistability is important as a basis for new strategies in organ transplantation and therapy.
PMCID: PMC3068929  PMID: 21483483
3.  Stimulation of mitochondrial proton conductance by hydroxynonenal requires a high membrane potential 
Bioscience Reports  2008;28(Pt 2):83-88.
Mild uncoupling of oxidative phosphorylation, caused by a leak of protons back into the matrix, limits mitochondrial production of ROS (reactive oxygen species). This proton leak can be induced by the lipid peroxidation products of ROS, such as HNE (4-hydroxynonenal). HNE activates uncoupling proteins (UCP1, UCP2 and UCP3) and ANT (adenine nucleotide translocase), thereby providing a negative feedback loop. The mechanism of activation and the conditions necessary to induce uncoupling by HNE are unclear. We have found that activation of proton leak by HNE in rat and mouse skeletal muscle mitochondria is dependent on incubation with respiratory substrate. In the presence of HNE, mitochondria energized with succinate became progressively more leaky to protons over time compared with mitochondria in the absence of either HNE or succinate. Energized mitochondria must attain a high membrane potential to allow HNE to activate uncoupling: a drop of 10–20 mV from the resting value is sufficient to blunt induction of proton leak by HNE. Uncoupling occurs through UCP3 (11%), ANT (64%) and other pathways (25%). Our findings have shown that exogenous HNE only activates uncoupling at high membrane potential. These results suggest that both endogenous HNE production and high membrane potential are required before mild uncoupling will be triggered to attenuate mitochondrial ROS production.
PMCID: PMC2518262  PMID: 18384278
adenine nucleotide translocase (ANT); 4-hydroxynonenal (HNE); mitochondria; proton leak; uncoupling protein 3 (UCP3); ANT, adenine nucleotide translocase; HNE, 4-hydroxynonenal; KO, knockout; ROS, reactive oxygen species; TPMP, triphenylmethylphosphonium; UCP, uncoupling protein; WT, wild-type.
4.  Mitochondrial Reactive Oxygen Species Production in Excitable Cells: Modulators of Mitochondrial and Cell Function 
Antioxidants & Redox Signaling  2009;11(6):1373-1414.
The mitochondrion is a major source of reactive oxygen species (ROS). Superoxide (O2•−) is generated under specific bioenergetic conditions at several sites within the electron-transport system; most is converted to H2O2 inside and outside the mitochondrial matrix by superoxide dismutases. H2O2 is a major chemical messenger that, in low amounts and with its products, physiologically modulates cell function. The redox state and ROS scavengers largely control the emission (generation scavenging) of O2•−. Cell ischemia, hypoxia, or toxins can result in excess O2•− production when the redox state is altered and the ROS scavenger systems are overwhelmed. Too much H2O2 can combine with Fe2+ complexes to form reactive ferryl species (e.g., Fe(IV) = O•). In the presence of nitric oxide (NO•), O2•− forms the reactant peroxynitrite (ONOO−), and ONOOH-induced nitrosylation of proteins, DNA, and lipids can modify their structure and function. An initial increase in ROS can cause an even greater increase in ROS and allow excess mitochondrial Ca2+ entry, both of which are factors that induce cell apoptosis and necrosis. Approaches to reduce excess O2•− emission include selectively boosting the antioxidant capacity, uncoupling of oxidative phosphorylation to reduce generation of O2•− by inducing proton leak, and reversibly inhibiting electron transport. Mitochondrial cation channels and exchangers function to maintain matrix homeostasis and likely play a role in modulating mitochondrial function, in part by regulating O2•− generation. Cell-signaling pathways induced physiologically by ROS include effects on thiol groups and disulfide linkages to modify posttranslationally protein structure to activate/inactivate specific kinase/phosphatase pathways. Hypoxia-inducible factors that stimulate a cascade of gene transcription may be mediated physiologically by ROS. Our knowledge of the role played by ROS and their scavenging systems in modulation of cell function and cell death has grown exponentially over the past few years, but we are still limited in how to apply this knowledge to develop its full therapeutic potential. Antioxid. Redox Signal. 11, 1373–1414.
Focus of review
Ambiguities about ROS
Overview of Mitochondrial Structure and Function
Mitochondrial Sources and Mechanism of ROS Generation
Requirement for charged membrane, electron flux, and O2
Reactive and nonreactive O2 species and reactants
Assessing ROS generation
Sites and conditions for mitochondrial ROS generation
Complex I (NADH ubiquinone oxidoreductase)
Complex III (co-enzyme Q, bc1 complex, ubiquinone/cytochrome c reductase)
Pathologic Induction of Mitochondrial ROS Release
ROS-induced ROS release
ROS-induced Ca2+ loading
ROS generation during tissue ischemia and hypoxia
Very low po2 and lack of mitochondrial ROS generation
Antioxidant Defenses Against Pathologic ROS Formation
SODs, catalase, cytochrome c, GSH, and TRXSH2, and other linked redox couples
Regulation of genes encoding mitochondrial antioxidant systems
ROS generation versus ROS scavenging
MPT pore opening and cytochrome c
Targets of Excess ROS Emission
DNA, proteins, and phospholipids
Role of cardiolipin
Approaches to Reduce Excess ROS
Capacity of mitochondrial and cell reductants
Exogenous SODs and catalase
Proton leak to modulate superoxide generation
Uncoupling proteins
HNE-induced proton leak
ROS-induced proton leak
Physiologic Modulation of Mitochondrial ROS Emission
H2O2 and ONOO− as chemical effectors
ROS modulation by cations
K+: A modulator of ROS generation?
Biphasic effect of KCa channels on ROS generation
KATP channel opening and ROS
Direct Ca2+-induced ROS unlikely
Rate of oxidative phosphorylation and ROS generation
Role of ROS in Triggering or Effecting Cardioprotection
Pathways and mechanisms
Inhibiting complex I and cardioprotection
Regulation of Cellular Processes by Mitochondria-Derived ROS
Cell signaling by oxidative modifications and redox systems
Examples of signaling by ROS
Importance of cysteine thiols in ROS-induced signaling
ROS oxidation reactions
O2 sensors
Hypoxia-inducible factors
ROS as O2 sensors
Peroxide-induced TCA shunts
Difficulties in understanding the role of ROS
Future directions
PMCID: PMC2842133  PMID: 19187004
5.  Localization of superoxide anion production to mitochondrial electron transport chain in 3-NPA-treated cells 
Mitochondrion  2006;6(5):235-244.
3-Nitropropionic acid (3-NPA), an inhibitor of succinate dehydrogenase (SDH) at complex II of the mitochondrial electron transport chain induces cellular energy deficit and oxidative stress-related neurotoxicity. In the present study, we identified the site of reactive oxygen species production in mitochondria. 3-NPA increased O2•− generation in mitochondria respiring on the complex I substrates pyruvate + malate, an effect fully inhibited by rotenone. Antimycin A increased O2•− production in the presence of complex I and/or II substrates. Addition of 3-NPA markedly increased antimycin A-induced O2•− production by mitochondria incubated with complex I substrates, but 3-NPA inhibited O2•− formation driven with the complex II substrate succinate. At 0.6 μM, myxothiazol inhibits complex III, but only partially decreases complex I activity, and allowed 3-NPA-induced O2•− formation; however, at 40 μM myxothiazol (which completely inhibits both complexes I and III) eliminated O2•− production from mitochondria respiring via complex I substrates. These results indicate that in the presence of 3-NPA, mitochondria generate O2•− from a site between the ubiquinol pool and the 3-NPA block in the respiratory complex II.
PMCID: PMC3031911  PMID: 17011837
3-NPA; Superoxide anion; Mitochondrial respiratory complexes
6.  Reversible inactivation of dihydrolipoamide dehydrogenase by mitochondrial hydrogen peroxide 
Free radical research  2012;47(2):123-133.
Under oxidative stress conditions, mitochondria are the major site for cellular production of reactive oxygen species (ROS) such as superoxide anion and H2O2 that can attack numerous mitochondrial proteins including dihydrolipoamide dehydrogenase (DLDH). While DLDH is known to be vulnerable to oxidative inactivation, the mechanisms have not been clearly elucidated. The present study was therefore designed to investigate the mechanisms of DLDH oxidative inactivation by mitochondrial reactive oxygen species (ROS). Mitochondria, isolated from rat brain, were incubated with mitochondrial respiratory substrates such as pyruvate/malate or succinate in the presence of electron transport chain inhibitors such as rotenone or antimycin A. This is followed by enzyme activity assay and gel-based proteomic analysis. The present study also examined whether ROS-induced DLDH oxidative inactivation could be reversed by reducing reagents such as DTT, cysteine, and glutathione. Results show that DLDH could only be inactivated by complex III- but not complex I-derived ROS; and the accompanying loss of activity due to the inactivation could be restored by cysteine and glutathione, indicating that DLDH oxidative inactivation by complex III-derived ROS was a reversible process. Further studies using catalase indicate that it was H2O2 instead of superoxide anion that was responsible for DLDH inactivation. Moreover, using sulfenic acid-specific labeling techniques in conjunction with two-dimensional Western blot analysis, we show that protein sulfenic acid formation (also known as sulfenation) was associated with the loss of DLDH enzymatic activity observed under our experimental conditions. Additionally, such oxidative modification was shown to be associated with preventing DLDH from further inactivation by the thiol-reactive reagent N-ethylmaleimide. Taken together, the present study provides insights into the mechanisms of DLDH oxidative inactivation by mitochondrial H2O2.
PMCID: PMC3690130  PMID: 23205777
brain; dihydrolipoamide dehydrogenase; H2O2; mitochondria; reactive oxygen species; reversible inactivation; sulfenic acid; sulfenation
7.  Superoxide Anion, Uncoupling Proteins and Alzheimer’s Disease 
Superoxide anion is the first generated reactive oxygen species (ROS) after oxygen enters living cells. It was once considered to be highly deleterious to cell functions and aging. Therefore, antioxidants were suggested to prevent aging and degenerative diseases. However, superoxide signaling has been shown in many physiological responses such as transcriptional regulation, protein activation, bioenergy output, cell proliferation and apoptosis. The uncoupling proteins (UCPs) are a family of mitochondrial anion-carrier proteins located in the inner mitochondrial membrane and are considered to reduce the generation of superoxide anion through the mitochondrial mild uncoupling. UCPs are important in prevention of mitochondrial excessive generation of ROS, transfer of mitochondrial substrates, mitochondrial calcium uniport and regulation of thermogenesis. Superoxide anion and uncoupling proteins are linked to Alzheimer’s disease in mitochondria. Simultaneous disorders of superoxide and uncoupling proteins create the conditions for neuronal oxidative damages. On the one hand, sustained oxidative damage causes neuronal apoptosis and eventually, accumulated neuronal apoptosis, leading to exacerbations of Alzheimer’s disease. On the other hand, our study has shown that UCP2 and UCP4 have important impact on mitochondrial calcium concentration of nerve cells, suggesting that their abnormal expression may involve in the pathogenesis of Alzheimer’s disease.
PMCID: PMC2872223  PMID: 20490313
superoxide anion; uncoupling protein; Alzheimer’s disease; oxidative stress; neurodegeneration
8.  Complex I generated, mitochondrial matrix-directed superoxide is released from the mitochondria through voltage dependent anion channels 
Mitochondrial complex I has previously been shown to release superoxide exclusively towards the mitochondrial matrix, whereas complex III releases superoxide to both the matrix and the cytosol. Superoxide produced at Complex III has been shown to exit the mitochondria through voltage dependent anion channels (VDAC). To test whether complex I-derived, mitochondrial matrix-directed superoxide can be released to the cytosol, we measured superoxide generation in mitochondria isolated from wild type and from mice genetically altered to be deficient in MnSOD activity (TnIFastCreSod2fl/fl). Under experimental conditions that produce superoxide primarily by complex I (glutamate/malate plus rotenone, GM+R), MnSOD-deficient mitochondria release ~4-fold more superoxide than mitochondria isolated from wild type mice. Exogenous CuZnSOD completely abolished the EPR-derived GM+R signal in mitochondria isolated from both genotypes, evidence that confirms mitochondrial superoxide release. Addition of the VDAC inhibitor DIDS significantly reduced mitochondrial superoxide release (~75%) in mitochondria from either genotype respiring on GM+R. Conversely, inhibition of potential inner membrane sites of superoxide exit, including the matrix face of the mitochondrial permeability transition pore and the inner membrane anion channel did not reduce mitochondrial superoxide release in the presence of GM+R in mitochondria isolated from either genotype. These data support the concept that complex I-derived mitochondrial superoxide release does indeed occur and that the majority of this release occurs through VDACs.
PMCID: PMC3400138  PMID: 22613204
mitochondria; superoxide; voltage dependent anion channels
9.  Semiquinone Radicals from Oxygenated Polychlorinated Biphenyls: Electron Paramagnetic Resonance Studies 
Chemical Research in Toxicology  2008;21(7):1359-1367.
Polychlorinated biphenyls (PCBs) can be oxygenated to form very reactive hydroquinone and quinone products. A guiding hypothesis in the PCB research community is that some of the detrimental health effects of some PCBs are a consequence of these oxygenated forms undergoing one-electron oxidation or reduction, generating semiquinone radicals (SQ•−). These radicals can enter into a futile redox cycle resulting in the formation of reactive oxygen species, that is, superoxide and hydrogen peroxide. Here, we examine some of the properties and chemistry of these semiquinone free radicals. Using electron paramagnetic resonance (EPR) to detect SQ•− formation, we observed that (i) xanthine oxidase can reduce quinone PCBs to the corresponding SQ•−; (ii) the heme-containing peroxidases (horseradish and lactoperoxidase) can oxidize hydroquinone PCBs to the corresponding SQ•−; (iii) tyrosinase acting on PCB ortho-hydroquinones leads to the formation of SQ•−; (iv) mixtures of PCB quinone and hydroquinone form SQ•− via a comproportionation reaction; (v) SQ•− are formed when hydroquinone-PCBs undergo autoxidation in high pH buffer (≈>pH 8); and, surprisingly, (vi) quinone-PCBs in high pH buffer can also form SQ•−; (vii) these observations along with EPR suggest that hydroxide anion can add to the quinone ring; (viii) H2O2 in basic solution reacts rapidly with PCB-quinones; and (ix) at near-neutral pH SOD can catalyze the oxidization of PCB-hydroquinone to quinone, yielding H2O2. However, using 5,5-dimethylpyrroline-1-oxide (DMPO) as a spin-trapping agent, we did not trap superoxide, indicating that generation of superoxide from SQ•− is not kinetically favorable. These observations demonstrate multiple routes for the formation of SQ•− from PCB-quinones and hydroquinones. Our data also point to futile redox cycling as being one mechanism by which oxygenated PCBs can lead to the formation of reactive oxygen species, but this is most efficient in the presence of SOD.
PMCID: PMC2740386  PMID: 18549251
10.  Human neuronal uncoupling proteins 4 and 5 (UCP4 and UCP5): structural properties, regulation, and physiological role in protection against oxidative stress and mitochondrial dysfunction 
Brain and Behavior  2012;2(4):468-478.
Uncoupling proteins (UCPs) belong to a large family of mitochondrial solute carriers 25 (SLC25s) localized at the inner mitochondrial membrane. UCPs transport protons directly from the intermembrane space to the matrix. Of five structural homologues (UCP1 to 5), UCP4 and 5 are principally expressed in the central nervous system (CNS). Neurons derived their energy in the form of ATP that is generated through oxidative phosphorylation carried out by five multiprotein complexes (Complexes I–V) embedded in the inner mitochondrial membrane. In oxidative phosphorylation, the flow of electrons generated by the oxidation of substrates through the electron transport chain to molecular oxygen at Complex IV leads to the transport of protons from the matrix to the intermembrane space by Complex I, III, and IV. This movement of protons to the intermembrane space generates a proton gradient (mitochondrial membrane potential; MMP) across the inner membrane. Complex V (ATP synthase) uses this MMP to drive the conversion of ADP to ATP. Some electrons escape to oxygen-forming harmful reactive oxygen species (ROS). Proton leakage back to the matrix which bypasses Complex V resulting in a major reduction in ROS formation while having a minimal effect on MMP and hence, ATP synthesis; a process termed “mild uncoupling.” UCPs act to promote this proton leakage as means to prevent excessive build up of MMP and ROS formation. In this review, we discuss the structure and function of mitochondrial UCPs 4 and 5 and factors influencing their expression. Hypotheses concerning the evolution of the two proteins are examined. The protective mechanisms of the two proteins against neurotoxins and their possible role in regulating intracellular calcium movement, particularly with regard to the pathogenesis of Parkinson's disease are discussed.
PMCID: PMC3432969  PMID: 22950050
Energy homeostasis; mitochondrial dysfunction; neurodegeneration; neuroprotection; oxidative stress; uncoupling proteins
11.  High membrane potential promotes alkenal-induced mitochondrial uncoupling and influences adenine nucleotide translocase conformation 
Biochemical Journal  2008;413(Pt 2):323-332.
Mitochondria generate reactive oxygen species, whose downstream lipid peroxidation products, such as 4-hydroxynonenal, induce uncoupling of oxidative phosphorylation by increasing proton leak through mitochondrial inner membrane proteins such as the uncoupling proteins and adenine nucleotide translocase. Using mitochondria from rat liver, which lack uncoupling proteins, in the present study we show that energization (specifically, high membrane potential) is required for 4-hydroxynonenal to activate proton conductance mediated by adenine nucleotide translocase. Prolonging the time at high membrane potential promotes greater uncoupling. 4-Hydroxynonenal-induced uncoupling via adenine nucleotide translocase is prevented but not readily reversed by addition of carboxyatractylate, suggesting a permanent change (such as adduct formation) that renders the translocase leaky to protons. In contrast with the irreversibility of proton conductance, carboxyatractylate added after 4-hydroxynonenal still inhibits nucleotide translocation, implying that the proton conductance and nucleotide translocation pathways are different. We propose a model to relate adenine nucleotide translocase conformation to proton conductance in the presence or absence of 4-hydroxynonenal and/or carboxyatractylate.
PMCID: PMC2474560  PMID: 18426390
carboxyatractylate (CAtr); 4-hydroxynonenal (HNE); proton leak; rat liver mitochondrion; trypsin; ANT, adenine nucleotide translocase; CAtr, carboxyatractylate; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; HNE, 4-hydroxynonenal; ROS, reactive oxygen species; TPMP, triphenylmethylphosphonium; UCP, uncoupling protein
12.  Salinomycin Effects on Mitochondrial Ion Translocation and Respiration 
The effects of salinomycin on alkali cation transport and membrane functions in rat liver mitochondria have been investigated. After potassium uptake, stimulated by valinomycin or monazomycin in the presence of adenosine 5′-triphosphate, salinomycin caused rapid release of K+ from mitochondria. Salinomycin reversed valinomycin- or monazomycin-induced oscillatory swelling of mitochondria preloaded with K+, Rb+, and Na+ but was without effect on Li+ or Cs+ preloaded mitochondria. Salinomycin blocked the retention of K+ more effectively than the retention of Rb+ or Na+. Salinomycin inhibited both coupled and uncoupled respiration with strict substrate specificity in medium of low but not in high K+ concentration. The oxidation of glutamate, α-ketoglutarate, and malate plus pyruvate was inhibited by salinomycin, but that of β-hydroxybutyrate or succinate was not significantly affected. Salinomycin inhibited adenosine triphosphatase activity of mitochondria induced by valinomycin or monazomycin in K+ and Rb+ medium without significantly affecting adenosine triphosphatase activity in Li+, Na+, or Cs+ medium. Oxidative phosphorylation in mitochondria was inhibited by salinomycin but the inhibitory effect of salinomycin lacked the substrate specificity observed for respiration. It is proposed that salinomycin perturbs mitochondrial functions by acting as a mobile carrier for alkali cations through membranes.
PMCID: PMC429593  PMID: 131509
13.  How mitochondria produce reactive oxygen species 
Biochemical Journal  2008;417(Pt 1):1-13.
The production of ROS (reactive oxygen species) by mammalian mitochondria is important because it underlies oxidative damage in many pathologies and contributes to retrograde redox signalling from the organelle to the cytosol and nucleus. Superoxide (O2•−) is the proximal mitochondrial ROS, and in the present review I outline the principles that govern O2•− production within the matrix of mammalian mitochondria. The flux of O2•− is related to the concentration of potential electron donors, the local concentration of O2 and the second-order rate constants for the reactions between them. Two modes of operation by isolated mitochondria result in significant O2•− production, predominantly from complex I: (i) when the mitochondria are not making ATP and consequently have a high Δp (protonmotive force) and a reduced CoQ (coenzyme Q) pool; and (ii) when there is a high NADH/NAD+ ratio in the mitochondrial matrix. For mitochondria that are actively making ATP, and consequently have a lower Δp and NADH/NAD+ ratio, the extent of O2•− production is far lower. The generation of O2•− within the mitochondrial matrix depends critically on Δp, the NADH/NAD+ and CoQH2/CoQ ratios and the local O2 concentration, which are all highly variable and difficult to measure in vivo. Consequently, it is not possible to estimate O2•− generation by mitochondria in vivo from O2•−-production rates by isolated mitochondria, and such extrapolations in the literature are misleading. Even so, the description outlined here facilitates the understanding of factors that favour mitochondrial ROS production. There is a clear need to develop better methods to measure mitochondrial O2•− and H2O2 formation in vivo, as uncertainty about these values hampers studies on the role of mitochondrial ROS in pathological oxidative damage and redox signalling.
PMCID: PMC2605959  PMID: 19061483
complex I; hydrogen peroxide; mitochondrion; reactive oxygen species (ROS); respiratory chain; superoxide; CoQ, coenzyme Q; CoQH2, reduced CoQ; DPI, diphenyleneiodonium; ETF, electron transfer flavoprotein; α-GPDH, α-glycerophosphate dehydrogenase; HIF-1, hypoxia-inducible factor-1; αKGDH, 2-oxoglutarate dehydrogenase (α-ketoglutarate dehydrogenase); PHD, prolyl hydroxylase; RET, reverse electron transport; ROS, reactive oxygen species; SMP, submitochondrial particle; SOD, superoxide dismutase
14.  Superoxide Flashes in Single Mitochondria 
Cell  2008;134(2):279-290.
The mitochondrion is the primary source of reactive oxygen species (ROS) in eukaryotic cells. With the aid of a novel mitochondrial matrix-targeted superoxide indicator, here we show that individual mitochondria undergo spontaneous bursts of superoxide generation, termed “superoxide flashes”. Superoxide flashes occur randomly in space and time, exhibit all-or-none properties, and reflect elementary events of superoxide production within single mitochondria across a wide diversity of cells. Individual flashes are triggered by transient openings of the mitochondrial permeability transition pore (mPTP) and are fueled by electron transfer complexes-dependent superoxide production. While decreased during cardiac hypoxia/anoxia, a flurry of superoxide flash activity contributes to the destructive rebound ROS burst observed during early reoxygenation after anoxia. The discovery of superoxide flashes reveals a novel mechanism for quantal ROS production by individual mitochondria and substantiates the central role of mPTP in oxidative stress related pathology in addition to its well-known role in apoptosis.
PMCID: PMC2547996  PMID: 18662543
15.  Mitochondrial Superoxide Dismutase - Signals of Distinction 
Mitochondrial superoxide dismutase (MnSOD) neutralizes the highly reactive superoxide radical (O2·−), the first member in a plethora of mitochondrial reactive oxygen species (ROS). Over the past decades, research has extended the prevailing view of mitochondrion well beyond the generation of cellular energy to include its importance in cell survival and cell death. In the normal state of a cell, endogenous antioxidant enzyme systems maintain the level of reactive oxygen species generated by the mitochondrial respiratory chain. Mammalian mitochondria are important to the production of reactive oxygen species, which underlie oxidative damage in many pathological conditions and contribute to retrograde redox signaling from the organelle to the cytosol and nucleus. Mitochondria are further implicated in various metabolic and aging-related diseases that are now postulated to be caused by misregulation of physiological systems rather than pure accumulation of oxidative damage. Thus, the signaling mechanisms within mitochondria, and between the organelle and its environment, have gained interest as potential drug targets. Here, we discuss redox events in mitochondria that lead to retrograde signaling, the role of redox events in disease, and their potential to serve as therapeutic targets.
PMCID: PMC3427752  PMID: 21355846
MnSOD; Retrograde signaling; Oxidative stress; Redox signaling; Apoptotic pathways; Oxidative modification; mtDNA; TOR signaling
16.  Identification of a novel mitochondrial uncoupler that does not depolarize the plasma membrane☆ 
Molecular Metabolism  2013;3(2):114-123.
Dysregulation of oxidative phosphorylation is associated with increased mitochondrial reactive oxygen species production and some of the most prevalent human diseases including obesity, cancer, diabetes, neurodegeneration, and heart disease. Chemical 'mitochondrial uncouplers' are lipophilic weak acids that transport protons into the mitochondrial matrix via a pathway that is independent of ATP synthase, thereby uncoupling nutrient oxidation from ATP production. Mitochondrial uncouplers also lessen the proton motive force across the mitochondrial inner membrane and thereby increase the rate of mitochondrial respiration while decreasing production of reactive oxygen species. Thus, mitochondrial uncouplers are valuable chemical tools that enable the measurement of maximal mitochondrial respiration and they have been used therapeutically to decrease mitochondrial reactive oxygen species production. However, the most widely used protonophore uncouplers such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and 2,4-dinitrophenol have off-target activity at other membranes that lead to a range of undesired effects including plasma membrane depolarization, mitochondrial inhibition, and cytotoxicity. These unwanted properties interfere with the measurement of mitochondrial function and result in a narrow therapeutic index that limits their usefulness in the clinic. To identify new mitochondrial uncouplers that lack off-target activity at the plasma membrane we screened a small molecule chemical library. Herein we report the identification and validation of a novel mitochondrial protonophore uncoupler (2-fluorophenyl){6-[(2-fluorophenyl)amino](1,2,5-oxadiazolo[3,4-e]pyrazin-5-yl)}amine, named BAM15, that does not depolarize the plasma membrane. Compared to FCCP, an uncoupler of equal potency, BAM15 treatment of cultured cells stimulates a higher maximum rate of mitochondrial respiration and is less cytotoxic. Furthermore, BAM15 is bioactive in vivo and dose-dependently protects mice from acute renal ischemic-reperfusion injury. From a technical standpoint, BAM15 represents an effective new tool that allows the study of mitochondrial function in the absence of off-target effects that can confound data interpretation. From a therapeutic perspective, BAM15-mediated protection from ischemia-reperfusion injury and its reduced toxicity will hopefully reignite interest in pharmacological uncoupling for the treatment of the myriad of diseases that are associated with altered mitochondrial function.
PMCID: PMC3953706  PMID: 24634817
ANT, adenine nucleotide translocase; ECAR, extracellular acidification rate; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; OCR, oxygen consumption rate; ROS, reactive oxygen species; TCA cycle, tricarboxylic acid cycle; TMPD, N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride; TMRM, tetramethylrhodamine; Mitochondria; Bioenergetics; FCCP; CCCP; DNP; Ischemia
17.  Cellular and Biochemical Actions of Melatonin which Protect Against Free Radicals: Role in Neurodegenerative Disorders 
Current Neuropharmacology  2008;6(3):203-214.
Molecular oxygen is toxic for anaerobic organisms but it is also obvious that oxygen is poisonous to aerobic organisms as well, since oxygen plays an essential role for inducing molecular damage. Molecular oxygen is a triplet radical in its ground-stage (.O-O.) and has two unpaired electrons that can undergoes consecutive reductions of one electron and generates other more reactive forms of oxygen known as free radicals and reactive oxygen species. These reactants (including superoxide radicals, hydroxyl radicals) possess variable degrees of toxicity.
Nitric oxide (NO•) contains one unpaired electron and is, therefore, a radical. NO• is generated in biological tissues by specific nitric oxide synthases and acts as an important biological signal. Excessive nitric oxide production, under pathological conditions, leads to detrimental effects of this molecule on tissues, which can be attributed to its diffusion-limited reaction with superoxide to form the powerful and toxic oxidant, peroxynitrite.
Reactive oxygen and nitrogen species are molecular “renegades”; these highly unstable products tend to react rapidly with adjacent molecules, donating, abstracting, or even sharing their outer orbital electron(s). This reaction not only changes the target molecule, but often passes the unpaired electron along to the target, generating a second free radical, which can then go on to react with a new target amplifying their effects.
This review describes the mechanisms of oxidative damage and its relationship with the most highly studied neurodegenerative diseases and the roles of melatonin as free radical scavenger and neurocytoskeletal protector.
PMCID: PMC2687933  PMID: 19506721
Melatonin; alzheimer; parkinson; oxidative stress; NO; neurodegeneration.
18.  Gossypol Inhibits Electron Transport and Stimulates ROS Generation in Yarrowia lipolytica Mitochondria 
This work studied the effect of gossypol on the mitochondrial respiratory chain of Yarrowia lipolytica. The compound was shown to inhibit mitochondrial electron transfer and stimulate generation of reactive oxygen species. The inhibition kinetics in oxidation of various substrates (NADH, succinate, α-glycerophosphate and pyruvate + malate) by isolated mitochondria was investigated. Analysis of the kinetic parameters showed gossypol to inhibit two fragments of the mitochondrial electron transfer chain: a) between coenzyme Q and cytochrome b with KIIIi of 118.3 μM (inhibition by the noncompetitive type), and b) at the level of exogenous NADH dehydrogenase with of KIi 17.2 μM (inhibition by the mixed type).
PMCID: PMC3315064  PMID: 22481982
Gossypol; ROS production; Kinetics of inhibition; Mitochondria; Yarrowia lipolytica.
19.  Opening of the MitoKATP Channel and Decoupling of Mitochondrial Complex II and III Contribute to the Suppression of Myocardial Reperfusion Hyperoxygenation 
Diazoxide, a mitochondrial ATP-sensitive potassium (mitoKATP) channel opener, protects the heart from ischemia-reperfusion injury. Diazoxide also inhibits mitochondrial complex II-dependent respiration in addition to its preconditioning effect. However, there are no prior studies of the role of diazoxide on post-ischemic myocardial oxygenation. In the current study, we determined the effect of diazoxide on the suppression of post-ischemic myocardial tissue hyperoxygenation in vivo, superoxide (O2−•) generation in isolated mitochondria, and impairment of the interaction between complex II and complex III in purified mitochondrial proteins. It was observed that diazoxide totally suppressed the post-ischemic myocardial hyperoxygenation. With succinate but not glutamate/malate as the substrate, diazoxide significantly increased ubisemiquinone-dependent O2−• generation, which was not blocked by 5-HD and glibenclamide. Using a model system, the super complex of succinate-cytochrome c reductase (SCR) hosting complex II and complex III, we also observed that diazoxide impaired complex II and its interaction with complex III with no effect on complex III. UV-visible spectral analysis revealed that diazoxide decreased succinate-mediated ferricytochrome b reduction in SCR. In conclusion, our results demonstrated that diazoxide suppressed the in vivo post-ischemic myocardial hyperoxygenation through opening the mitoKATP channel and ubisemiquinone-dependent O2−• generation via inhibiting mitochondrial complex II-dependent respiration.
PMCID: PMC3738814  PMID: 19851835
Mitochondria; Diazoxide; Superoxide; Ischemia Reperfusion; Oxygenation
20.  Generation of superoxide and singlet oxygen from a-tocopherolquinone and analogues 
Free radical research  2007;41(6):730-737.
Three potential routes to generation of reactive oxygen species from α-tocopherolquinone have been identified. The quinone of the water-soluble vitamin E analogue Trolox C (Trol-Q) is reduced by hydrated electron and isopropanol α-hydroxyalkyl radical, and the resulting semiquinone reacts with molecular oxygen to form superoxide with a second order rate constant of 1.3 × 108 dm3 mol−1 s−1, illustrating the potential for redox cycling. Illumination (UV-A, 355 nm) of the quinone of 2,2,5,7,8-pentamethyl-6-hydroxychromanol (PMHC-Q) leads to a reactive short-lived (ca 10−6 s) triplet state, able to oxidise tryptophan with a second order rate constant greater than 109 dm3 mol−1 s−1. The triplet states of these quinones sensitize singlet oxygen formation with quantum yields of about 0.8. Such potentially damaging reactions of α-tocopherolquinone may in part account for the recent findings that high levels of dietary vitamin E supplementation lack any beneficial effect and may lead to slightly enhanced levels of overall mortality.
PMCID: PMC2080821  PMID: 17516246
Tocopherolquinone; radical; superoxide; singlet oxygen; redox cycling; triplet
21.  Mitochondrial Dysfunction in Diabetes: From Molecular Mechanisms to Functional Significance and Therapeutic Opportunities 
Antioxidants & Redox Signaling  2010;12(4):537-577.
Given their essential function in aerobic metabolism, mitochondria are intuitively of interest in regard to the pathophysiology of diabetes. Qualitative, quantitative, and functional perturbations in mitochondria have been identified and affect the cause and complications of diabetes. Moreover, as a consequence of fuel oxidation, mitochondria generate considerable reactive oxygen species (ROS). Evidence is accumulating that these radicals per se are important in the pathophysiology of diabetes and its complications. In this review, we first present basic concepts underlying mitochondrial physiology. We then address mitochondrial function and ROS as related to diabetes. We consider different forms of diabetes and address both insulin secretion and insulin sensitivity. We also address the role of mitochondrial uncoupling and coenzyme Q. Finally, we address the potential for targeting mitochondria in the therapy of diabetes. Antioxid. Redox Signal. 12, 537–577.
Basic Physiology
Electron transport
Reactive oxygen species and mitochondria
Mitochondrial nitric oxide
Role of calcium and the mitochondrial permeability transition pore
Assessing Mitochondrial Function
Respiration and potential
ATP production and the proton leak
ROS production by isolated mitochondria
Site specificity of mitochondrial superoxide production
Mitochondrial ROS production in intact cells
Oxidative damage to mitochondria in intact cells
Mitochondrial Metabolism and Diabetes
General considerations
Mitochondrial diabetes
Type 1 and type 2 diabetes
Mitochondrial number and morphology
Mitochondrial biogenesis
Mitochondrial function in type 2 diabetes and insulin-resistant states
Is mitochondrial impairment a cause of insulin resistance?
Mitochondrial respiratory coupling and insulin release
Mitochondrial function in insulin-deficient diabetes
Diabetes and mitochondrial function in non–insulin-sensitive tissues
Mitochondria and cell-fuel selectivity
Diabetic cardiomyopathy and mitochondrial function
Mitochondrial ROS and Diabetes
ROS production and the cause of diabetes
Oxidative damage and pancreatic islet β cells
ROS and oxidative damage in insulin-sensitive target tissues
ROS and the complications of diabetes
Non–insulin-sensitive tissues (retina, renal, neural cells)
ROS and vascular cells
Mitochondrial Membrane Potential and Diabetes
Role of uncoupling proteins
Does membrane potential actually protect against superoxide production?
Coenzyme Q and Diabetes
Therapeutic Implications
Improving mitochondrial metabolism
Lifestyle modification
Pharmacologic intervention
Controlling ROS production and oxidative damage
Mitochondria-targeted antioxidants
Metabolic effects of mitochondria-targeted antioxidants
Mitochondria-targeted antioxidant peptides
Targeting superoxide
PMCID: PMC2824521  PMID: 19650713
22.  MICU1 is an Essential Gatekeeper for MCU-Mediated Mitochondrial Ca2+ Uptake That Regulates Cell Survival 
Cell  2012;151(3):630-644.
Mitochondrial Ca2+ (Ca2+m) uptake is mediated by an inner membrane Ca2+ channel called the uniporter. Ca2+ uptake is driven by the considerable voltage present across the inner membrane (ΔΨm) generated by proton pumping by the respiratory chain. Mitochondrial matrix Ca2+ concentration is maintained 5–6 orders of magnitude lower than its equilibrium level, but the molecular mechanisms for how this is achieved are not clear. Here we demonstrate that the mitochondrial protein MICU1 is required to preserve normal [Ca2+]m under basal conditions. In its absence, mitochondria become constitutively loaded with Ca2+, triggering excessive reactive oxygen species generation and sensitivity to apoptotic stress. MICU1 interacts with the uniporter pore-forming subunit MCU and sets a Ca2+ threshold for Ca2+m uptake without affecting the kinetic properties of MCU-mediated Ca2+ uptake. Thus, MICU1 is a gatekeeper of MCU-mediated Ca2+m uptake that is essential to prevent [Ca2+]m overload and associated stress.
PMCID: PMC3486697  PMID: 23101630
23.  Elucidation of the effects of lipoperoxidation on the mitochondrial electron transport chain using yeast mitochondria with manipulated fatty acid content 
Lipoperoxidative damage to the respiratory chain proteins may account for disruption in mitochondrial electron transport chain (ETC) function and could lead to an augment in the production of reactive oxygen species (ROS). To test this hypothesis, we investigated the effects of lipoperoxidation on ETC function and cytochromes spectra of Saccharomyces cerevisiae mitochondria. We compared the effects of Fe2+ treatment on mitochondria isolated from yeast with native (lipoperoxidation-resistant) and modified (lipoperoxidation-sensitive) fatty acid composition. Augmented sensitivity to oxidative stress was observed in the complex III-complex IV segment of the ETC. Lipoperoxidation did not alter the cytochromes content. Under lipoperoxidative conditions, cytochrome c reduction by succinate was almost totally eliminated by superoxide dismutase and stigmatellin. Our results suggest that lipoperoxidation impairs electron transfer mainly at cytochrome b in complex III, which leads to increased resistance to antimycin A and ROS generation due to an electron leak at the level of the QO site of complex III.
PMCID: PMC2922399  PMID: 19224349
Lipoperoxidation; Cytochromes; Yeast mitochondria; Iron; Electron transport chain
24.  Mitochondria are the primary source of the H2O2 signal for glucocorticoid-induced apoptosis of lymphoma cells 
Glucocorticoids are a class of steroid hormones commonly used for the treatment of hematological malignancies due to their ability to induce apoptosis in lymphoid cells. An understanding of the critical steps in glucocorticoid-induced apoptosis is required to identify sources of drug resistance. Previously, we found that an increase in hydrogen peroxide is a necessary signal for glucocorticoid-induced apoptosis. In the current study, we found that mitochondria are the source of the signal. Glucocorticoid treatment inhibited Complex I and Complex III of the electron transport chain (ETC). Mitochondrial matrix reactive oxygen species (ROS) increased concomitantly with the oxidation of the mitochondrial glutathione pool. Treatment with Tiron, a superoxide scavenger, inhibited the signal. This suggests that the hydrogen peroxide signal originates as superoxide from the mitochondria and is metabolized to hydrogen peroxide. An inability to generate mitochondrial oxidants in response to glucocorticoids could cause drug resistance.
PMCID: PMC3404723  PMID: 22844350
apoptosis; lymphoma mitochondria; reactive oxygen species
25.  Mitochondria are the primary source of the H2O2 signal for glucocorticoid-induced apoptosis of lymphoma cells 
Glucocorticoids are a class of steroid hormones commonly used for the treatment of hematological malignancies due to their ability to induce apoptosis in lymphoid cells. An understanding of the critical steps in glucocorticoid-induced apoptosis is required to identify sources of drug resistance. Previously, we found that an increase in hydrogen peroxide is a necessary signal for glucocorticoid-induced apoptosis. In the current study, we found that mitochondria are the source of the signal. Glucocorticoid treatment inhibited Complex I and Complex III of the electron transport chain (ETC). Mitochondrial matrix reactive oxygen species (ROS) increased concomitantly with the oxidation of the mitochondrial glutathione pool. Treatment with Tiron, a superoxide scavenger, inhibited the signal. This suggests that the hydrogen peroxide signal originates as superoxide from the mitochondria and is metabolized to hydrogen peroxide. An inability to generate mitochondrial oxidants in response to glucocorticoids could cause drug resistance.
PMCID: PMC3404723  PMID: 22844350
apoptosis; lymphoma mitochondria; reactive oxygen species

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