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1.  Multistationary and Oscillatory Modes of Free Radicals Generation by the Mitochondrial Respiratory Chain Revealed by a Bifurcation Analysis 
PLoS Computational Biology  2012;8(9):e1002700.
The mitochondrial electron transport chain transforms energy satisfying cellular demand and generates reactive oxygen species (ROS) that act as metabolic signals or destructive factors. Therefore, knowledge of the possible modes and bifurcations of electron transport that affect ROS signaling provides insight into the interrelationship of mitochondrial respiration with cellular metabolism. Here, a bifurcation analysis of a sequence of the electron transport chain models of increasing complexity was used to analyze the contribution of individual components to the modes of respiratory chain behavior. Our algorithm constructed models as large systems of ordinary differential equations describing the time evolution of the distribution of redox states of the respiratory complexes. The most complete model of the respiratory chain and linked metabolic reactions predicted that condensed mitochondria produce more ROS at low succinate concentration and less ROS at high succinate levels than swelled mitochondria. This prediction was validated by measuring ROS production under various swelling conditions. A numerical bifurcation analysis revealed qualitatively different types of multistationary behavior and sustained oscillations in the parameter space near a region that was previously found to describe the behavior of isolated mitochondria. The oscillations in transmembrane potential and ROS generation, observed in living cells were reproduced in the model that includes interaction of respiratory complexes with the reactions of TCA cycle. Whereas multistationarity is an internal characteristic of the respiratory chain, the functional link of respiration with central metabolism creates oscillations, which can be understood as a means of auto-regulation of cell metabolism.
Author Summary
The mitochondrial respiratory chain shows a variety of modes of behavior. In living cells, flashes of ROS production and oscillations accompanied by a decrease of transmembrane potential can be registered. The mechanisms of such complex behavior are difficult to rationalize without a mathematical formalization of mitochondrial respiration. Our most complete model of mitochondrial respiration accounts for the details of electron transport, reproducing the observed types of behavior, which includes the existence of multiple steady states and periodic oscillations. This most detailed model contains hundreds of differential equations, and such complexity makes it difficult to grasp the main determinants of its behavior. Therefore the full model was reduced to a simplified description of complex III only, and numerical bifurcation analysis was used to study its behavior. Then the evolution of its behavior was traced in a sequence of models with increasing complexity leading back to the full model. This analysis revealed the mechanism of switching between the modes of behavior and the conditions for persistence in a given state, which defines ATP production, ROS signaling and destructive effects. This is important for understanding the biochemical basics of many systemic diseases.
doi:10.1371/journal.pcbi.1002700
PMCID: PMC3447950  PMID: 23028295
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.
doi:10.1371/journal.pcbi.1001115
PMCID: PMC3068929  PMID: 21483483
3.  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.
doi:10.1016/j.mito.2006.07.008
PMCID: PMC3031911  PMID: 17011837
3-NPA; Superoxide anion; Mitochondrial respiratory complexes
4.  Reactive Oxygen Species Production in Energized Cardiac Mitochondria During Hypoxia/Reoxygenation: Modulation by Nitric Oxide 
Circulation research  2008;103(8):873-880.
Mitochondria are an important source of reactive oxygen species (ROS) implicated in ischemia/reperfusion injury. When isolated from ischemic myocardium, mitochondria demonstrate increased ROS production as a result of damage to electron transport complexes. To investigate the mechanisms, we studied effects of hypoxia/reoxygenation on ROS production by isolated energized heart mitochondria. ROS production, tracked using Fe2+-catalyzed, H2O2-dependent H2DCF oxidation or Amplex Red, was similar during normoxia and hypoxia, but markedly increased during reoxygenation, in proportion to the duration of hypoxia. In contrast, if mitochondria were rapidly converted from normoxia to near-anoxia ([O2]< 1 μM), the increase in H2DCF oxidation rate during reoxygenation was markedly blunted. To elicit the robust increase in H2DCF oxidation rate during reoxygenation, hypoxia had to be severe enough to cause partial, but not complete, respiratory chain inhibition (as shown by partial dissipation of membrane potential and increased NADH auto-fluorescence). Consistent with its cardioprotective actions, nitric oxide (•NO) abrogated increased H2DCF oxidation under these conditions, as well as attenuating ROS-induced increases in matrix [Fe2+] and aconitase inhibition caused by antimycin. Collectively, these results suggest that a) hypoxia sufficient to cause partial respiratory inhibition is more damaging to mitochondria than near-anoxia; b) •NO suppresses ROS-induced damage to electron transport complexes, probably by forming •NO-Fe2+ complexes in the presence of glutathione which inhibit hydroxyl radical formation.
doi:10.1161/CIRCRESAHA.108.180869
PMCID: PMC2755534  PMID: 18776040
Mitochondria; Reactive Oxygen Species; Hypoxia/Reoxygenation; Nitric Oxide
5.  Mitochondrial Dysfunction Increases Oxidative Stress and Decreases Chronological Life Span in Fission Yeast 
PLoS ONE  2008;3(7):e2842.
Background
Oxidative stress is a probable cause of aging and associated diseases. Reactive oxygen species (ROS) originate mainly from endogenous sources, namely the mitochondria.
Methodology/Principal Findings
We analyzed the effect of aerobic metabolism on oxidative damage in Schizosaccharomyces pombe by global mapping of those genes that are required for growth on both respiratory-proficient media and hydrogen-peroxide-containing fermentable media. Out of a collection of approximately 2700 haploid yeast deletion mutants, 51 were sensitive to both conditions and 19 of these were related to mitochondrial function. Twelve deletion mutants lacked components of the electron transport chain. The growth defects of these mutants can be alleviated by the addition of antioxidants, which points to intrinsic oxidative stress as the origin of the phenotypes observed. These respiration-deficient mutants display elevated steady-state levels of ROS, probably due to enhanced electron leakage from their defective transport chains, which compromises the viability of chronologically-aged cells.
Conclusion/Significance
Individual mitochondrial dysfunctions have often been described as the cause of diseases or aging, and our global characterization emphasizes the primacy of oxidative stress in the etiology of such processes.
doi:10.1371/journal.pone.0002842
PMCID: PMC2475502  PMID: 18665268
6.  Oxidation of Fatty Acids Is the Source of Increased Mitochondrial Reactive Oxygen Species Production in Kidney Cortical Tubules in Early Diabetes 
Diabetes  2012;61(8):2074-2083.
Mitochondrial reactive oxygen species (ROS) cause kidney damage in diabetes. We investigated the source and site of ROS production by kidney cortical tubule mitochondria in streptozotocin-induced type 1 diabetes in rats. In diabetic mitochondria, the increased amounts and activities of selective fatty acid oxidation enzymes is associated with increased oxidative phosphorylation and net ROS production with fatty acid substrates (by 40% and 30%, respectively), whereas pyruvate oxidation is decreased and pyruvate-supported ROS production is unchanged. Oxidation of substrates that donate electrons at specific sites in the electron transport chain (ETC) is unchanged. The increased maximal production of ROS with fatty acid oxidation is not affected by limiting the electron flow from complex I into complex III. The maximal capacity of the ubiquinol oxidation site in complex III in generating ROS does not differ between the control and diabetic mitochondria. In conclusion, the mitochondrial ETC is neither the target nor the site of ROS production in kidney tubule mitochondria in short-term diabetes. Mitochondrial fatty acid oxidation is the source of the increased net ROS production, and the site of electron leakage is located proximal to coenzyme Q at the electron transfer flavoprotein that shuttles electrons from acyl-CoA dehydrogenases to coenzyme Q.
doi:10.2337/db11-1437
PMCID: PMC3402323  PMID: 22586586
7.  Mitochondrial Dysfunction in Diabetes: From Molecular Mechanisms to Functional Significance and Therapeutic Opportunities 
Antioxidants & Redox Signaling  2010;12(4):537-577.
Abstract
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.
Introduction
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
Fission/fusion
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
Summary
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
Summary
Mitochondrial Membrane Potential and Diabetes
Role of uncoupling proteins
Does membrane potential actually protect against superoxide production?
Summary
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
Summary
doi:10.1089/ars.2009.2531
PMCID: PMC2824521  PMID: 19650713
8.  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.
doi:10.1007/s10863-009-9200-3
PMCID: PMC2922399  PMID: 19224349
Lipoperoxidation; Cytochromes; Yeast mitochondria; Iron; Electron transport chain
9.  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.
doi:10.3109/10715762.2012.752078
PMCID: PMC3690130  PMID: 23205777
brain; dihydrolipoamide dehydrogenase; H2O2; mitochondria; reactive oxygen species; reversible inactivation; sulfenic acid; sulfenation
10.  Mitochondrial Dysfunction Promotes Breast Cancer Cell Migration and Invasion through HIF1α Accumulation via Increased Production of Reactive Oxygen Species 
PLoS ONE  2013;8(7):e69485.
Although mitochondrial dysfunction has been observed in various types of human cancer cells, the molecular mechanism underlying mitochondrial dysfunction mediated tumorigenesis remains largely elusive. To further explore the function of mitochondria and their involvement in the pathogenic mechanisms of cancer development, mitochondrial dysfunction clones of breast cancer cells were generated by rotenone treatment, a specific inhibitor of mitochondrial electron transport complex I. These clones were verified by mitochondrial respiratory defect measurement. Moreover, those clones exhibited increased reactive oxygen species (ROS), and showed higher migration and invasive behaviors compared with their parental cells. Furthermore, antioxidant N-acetyl cysteine, PEG-catalase, and mito-TEMPO effectively inhibited cell migration and invasion in these clones. Notably, ROS regulated malignant cellular behavior was in part mediated through upregulation of hypoxia-inducible factor-1 α and vascular endothelial growth factor. Our results suggest that mitochondrial dysfunction promotes cancer cell motility partly through HIF1α accumulation mediated via increased production of reactive oxygen species.
doi:10.1371/journal.pone.0069485
PMCID: PMC3726697  PMID: 23922721
11.  Hypoxia-Adaptation Involves Mitochondrial Metabolic Depression and Decreased ROS Leakage 
PLoS ONE  2012;7(5):e36801.
Through long-term laboratory selection, we have generated a Drosophila melanogaster population that tolerates severe, normally lethal, level of hypoxia. This strain lives perpetually under severe hypoxic conditions (4% O2). In order to shed light on the mechanisms involved in this adaptation, we studied the respiratory function of isolated mitochondria from the thorax of hypoxia-adapted flies (AF) using polarographic oxygen consumption while monitoring superoxide generation by electron paramagnetic resonance (EPR) techniques. AF mitochondria exhibited a significant 30% decrease in respiratory rate during state 3, while enhancing the resting respiratory rate during State 4-oligo by 220%. The activity of individual electron transport complexes I, II and III were 107%, 65%, and 120% in AF mitochondria as compared to those isolated from control flies. The sharp decrease in complex II activity and modest increase in complexes I and III resulted in >60% reduction in superoxide leakage from AF mitochondria during both NAD+-linked state 3 and State 4-oligo respirations. These results provide evidence that flies with mitochondria exhibiting decreased succinate dehydrogenase activity and reduced superoxide leakage give flies an advantage for survival in long-term hypoxia.
doi:10.1371/journal.pone.0036801
PMCID: PMC3344937  PMID: 22574227
12.  Redox Regulation of Mitochondrial Function 
Antioxidants & Redox Signaling  2012;16(11):1323-1367.
Abstract
Redox-dependent processes influence most cellular functions, such as differentiation, proliferation, and apoptosis. Mitochondria are at the center of these processes, as mitochondria both generate reactive oxygen species (ROS) that drive redox-sensitive events and respond to ROS-mediated changes in the cellular redox state. In this review, we examine the regulation of cellular ROS, their modes of production and removal, and the redox-sensitive targets that are modified by their flux. In particular, we focus on the actions of redox-sensitive targets that alter mitochondrial function and the role of these redox modifications on metabolism, mitochondrial biogenesis, receptor-mediated signaling, and apoptotic pathways. We also consider the role of mitochondria in modulating these pathways, and discuss how redox-dependent events may contribute to pathobiology by altering mitochondrial function. Antioxid. Redox Signal. 16, 1323–1367.
I. Introduction
II. Cellular ROS, Redox, and Antioxidant Systems
A. Sources of cellular ROS
B. Mitochondrial generation of ROS
C. Redox and antioxidant systems
1. SODs
2. Catalase and NADPH
3. GPx, reduced glutathione, Grxs, and glutathione reductase
4. Prxs, Trx, and TrxR
5. Cytosolic and mitochondrial NADPH
D. Redox-active Cys
1. Cys oxidation states and functional modification of protein thiols
2. Protein S-glutathiolation
3. Protein S-nitrosation
4. Mitochondrial function and the thiol redox state
5. Mitochondrial disulfide-relay system
E. Metabolism, NADH/NAD+, and NADPH/NADP+
1. Regulation of glycolysis
2. SIRT, NAD+, and metabolic regulation
III. Functional Consequences of Redox Modifiers
A. NO·
1. Apoptosis and NO·
2. NO· and mitochondrial fission and fusion
3. NO· and mitochondrial respiration
B. Reactive oxygen species
1. NADPH activation of mitochondrial ROS
2. Other ROS-mediated ROS generators
3. Extracellular redox state and mitochondrial function
4. Mitochondrial ROS and receptor-mediated signaling
5. Mitochondrial respiration, inner mitochondrial membrane potential, and ROS
6. UCPs and mitochondrial function
IV. Redox Regulation of Mitochondrial Turnover
A. Mitochondrial biogenesis
1. Overview of biogenesis
2. ROS and biogenesis
3. Telomere dysfunction and mitochondrial biogenesis
B. ROS and mitophagy versus apoptosis
C. Heme oxygenase, carbon monoxide, and mitochondrial function
V. Redox Regulation of Apoptosis
A. ASK1 and Trx-mediated regulation of JNK
B. MPT pore and cell death
1. Mitochondrial permeability transition
2. Mitochondrial Ca2+, redox, and mitochondrial dysfunction
3. STAT3 and mitochondrial function
C. Lipid oxidation and apoptotic signaling
1. Cardiolipin oxidation and apoptosis
2. Membrane phospholipids and AIF-mediated cell death
VI. Mitochondrial Responses to Hypoxia
VII. Conclusion
doi:10.1089/ars.2011.4123
PMCID: PMC3324814  PMID: 22146081
13.  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
14.  Redox regulation of mitochondrial function with emphasis on cysteine oxidation reactions☆ 
Redox Biology  2013;2:123-139.
Mitochondria have a myriad of essential functions including metabolism and apoptosis. These chief functions are reliant on electron transfer reactions and the production of ATP and reactive oxygen species (ROS). The production of ATP and ROS are intimately linked to the electron transport chain (ETC). Electrons from nutrients are passed through the ETC via a series of acceptor and donor molecules to the terminal electron acceptor molecular oxygen (O2) which ultimately drives the synthesis of ATP. Electron transfer through the respiratory chain and nutrient oxidation also produces ROS. At high enough concentrations ROS can activate mitochondrial apoptotic machinery which ultimately leads to cell death. However, if maintained at low enough concentrations ROS can serve as important signaling molecules. Various regulatory mechanisms converge upon mitochondria to modulate ATP synthesis and ROS production. Given that mitochondrial function depends on redox reactions, it is important to consider how redox signals modulate mitochondrial processes. Here, we provide the first comprehensive review on how redox signals mediated through cysteine oxidation, namely S-oxidation (sulfenylation, sulfinylation), S-glutathionylation, and S-nitrosylation, regulate key mitochondrial functions including nutrient oxidation, oxidative phosphorylation, ROS production, mitochondrial permeability transition (MPT), apoptosis, and mitochondrial fission and fusion. We also consider the chemistry behind these reactions and how they are modulated in mitochondria. In addition, we also discuss emerging knowledge on disorders and disease states that are associated with deregulated redox signaling in mitochondria and how mitochondria-targeted medicines can be utilized to restore mitochondrial redox signaling.
Highlights
•Mitochondria are essential for various cellular processes.•Mitochondria are major sites for regulation by redox signaling.•Redox signaling regulates mitochondrial metabolism and function.•Redox signaling in mitochondria is deregulated in various disease states.
doi:10.1016/j.redox.2013.12.011
PMCID: PMC3895620  PMID: 24455476
Redox; S-glutathionylation; S-oxidation; S-nitrosylation; Mitochondria
15.  Differential Contribution of the Mitochondrial Respiratory Chain Complexes to Reactive Oxygen Species Production by Redox Cycling Agents Implicated in Parkinsonism 
Toxicological Sciences  2009;112(2):427-434.
Exposure to environmental pesticides can cause significant brain damage and has been linked with an increased risk of developing neurodegenerative disorders, including Parkinson's disease. Bipyridyl herbicides, such as paraquat (PQ), diquat (DQ), and benzyl viologen (BV), are redox cycling agents known to exert cellular damage through the production of reactive oxygen species (ROS). We examined the involvement of the mitochondrial respiratory chain in ROS production by bipyridyl herbicides. In isolated rat brain mitochondria, H2O2 production occurred with the following order of potency: BV > DQ > PQ in accordance with their measured ability to redox cycle. H2O2 production was significantly attenuated in all cases by antimycin A, an inhibitor of complex III. Interestingly, at micromolar (≤ 300μM) concentrations, PQ-induced H2O2 production was unaffected by complex I inhibition via rotenone, whereas DQ-induced H2O2 production was equally attenuated by inhibition of complex I or III. Moreover, complex I inhibition decreased BV-induced H2O2 production to a greater extent than with PQ or DQ. These data suggest that multiple sites within the respiratory chain contribute to H2O2 production by redox cycling bipyridyl herbicides. In primary midbrain cultures, H2O2 differed slightly with the following order of potency: DQ > BV > PQ. In this model, inhibition of complex III resulted in roughly equivalent inhibition of H2O2 production with all three compounds. These data identify a novel role for complex III dependence of mitochondrial ROS production by redox cycling herbicides, while emphasizing the importance of identifying mitochondrial mechanisms by which environmental agents generate oxidative stress contributing to parkinsonism.
doi:10.1093/toxsci/kfp223
PMCID: PMC2777080  PMID: 19767442
paraquat; redox cycling; mitochondria; Parkinson's disease
16.  Superoxide Flashes in Single Mitochondria 
Cell  2008;134(2):279-290.
SUMMARY
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.
doi:10.1016/j.cell.2008.06.017
PMCID: PMC2547996  PMID: 18662543
17.  Redox Regulation of Calcium Signaling in Cancer Cells by Ascorbic Acid Involving the Mitochondrial Electron Transport Chain 
Journal of Biophysics  2012;2012:921653.
Previously, we have reported that ascorbic acid regulates calcium signaling in human larynx carcinoma HEp-2 cells. To evaluate the precise mechanism of Ca2+ release by ascorbic acid, the effects of specific inhibitors of the electron transport chain components on mitochondrial reactive oxygen species (ROS) production and Ca2+ mobilization in HEp-2 cells were investigated. It was revealed that the mitochondrial complex III inhibitor (antimycin A) amplifies ascorbate-induced Ca2+ release from intracellular stores. The mitochondrial complex I inhibitor (rotenone) decreases Ca2+ release from intracellular stores in HEp-2 cells caused by ascorbic acid and antimycin A. In the presence of rotenone, antimycin A stimulates ROS production by mitochondria. Ascorbate-induced Ca2+ release in HEp-2 cells is shown to be unaffected by catalase. The results obtained suggest that Ca2+ release in HEp-2 cells caused by ascorbic acid is associated with induced mitochondrial ROS production. The data obtained are in line with the concept of redox signaling that explains oxidant action by compartmentalization of ROS production and oxidant targets.
doi:10.1155/2012/921653
PMCID: PMC3512304  PMID: 23227042
18.  Inhibition of Mitochondrial Respiration and Rapid Depletion of Mitochondrial Glutathione by β-Phenethyl Isothiocyanate: Mechanisms for Anti-Leukemia Activity 
Antioxidants & Redox Signaling  2011;15(12):2911-2921.
Abstract
Aims
β-Phenethyl isothiocyanate (PEITC) is a natural product with potent anticancer activity against human leukemia cells including drug-resistant primary leukemia cells from patients. This study aimed at investigating the key mechanisms that contribute to the potent anti-leukemia activity of PEITC and at evaluating its therapeutic potential.
Results
Our study showed that PEITC caused a rapid depletion of mitochondrial glutathione (GSH) and a significant elevation of reactive oxygen species (ROS) and nitric oxide, and induced a disruption of the mitochondrial electron transport complex I manifested by an early degradation of NADH dehydrogenase Fe-S protein-3 and a significant suppression of mitochondrial respiration. Using biochemical and pharmacological approaches, we further showed that inhibition of mitochondrial respiration alone by rotenone caused only a moderate cytotoxicity in leukemia cells, whereas a combination of respiratory inhibition and an ROS-generating agent exhibited a synergistic effect against leukemia and lymphoma cells.
Innovation and Conclusion
Although PEITC is a reactive compound and might have multiple mechanisms of action, we showed that a rapid depletion of GSH and inhibition of mitochondrial respiration are two important early events that induced synergistic cytotoxicity in leukemia cells. These findings not only suggest that PEITC is a promising compound for potential use in leukemia treatment, but also provide a basis for developing new therapeutic strategies to effectively kill leukemia cells by using a novel combination to modulate ROS and inhibit mitochondrial respiration. Antioxid. Redox Signal. 15, 2911–2921.
doi:10.1089/ars.2011.4170
PMCID: PMC3201634  PMID: 21827296
19.  The pro-oxidant chromium(VI) inhibits mitochondrial complex I, complex II, and aconitase in the bronchial epithelium: EPR markers for Fe-S proteins 
Free radical biology & medicine  2010;49(12):1903-1915.
Hexavalent chromium [Cr(VI)] compounds (e.g. chromates) are strong oxidants that readily enter cells where they are reduced to reactive Cr species that also facilitate reactive oxygen species (ROS) generation. Recent studies demonstrated inhibition and oxidation of the thioredoxin system, with greater effects on mitochondrial thioredoxin (Trx2). This implies that Cr(VI)-induced oxidant stress may be especially directed at the mitochondria. Examination of other redox-sensitive mitochondrial functions showed that Cr(VI) treatments that cause Trx2 oxidation in human bronchial epithelial cells also result in pronounced and irreversible inhibition of aconitase, a TCA cycle enzyme that has an iron-sulfur (Fe-S) center that is labile with respect to certain oxidants. The activities of electron transport complexes I and II were also inhibited, whereas complex III was not. Electron paramagnetic resonance (EPR) studies of samples at liquid helium temperature (10 K) showed a strong signal at g = 1.94 that is consistent with the inhibition of electron flow through complexes I and/or II. A signal at g = 2.02 was also observed which is consistent with oxidation of the Fe-S center of aconitase. The g = 1.94 signal was particularly intense and remained after extracellular Cr(VI) was removed, whereas the g = 2.02 signal declined in intensity after Cr(VI) was removed. A similar inhibition of these activities and analogous EPR findings were noted in bovine airways treated ex vivo with Cr(VI). Overall, the data support the hypothesis that Cr(VI) exposure has deleterious effects on a number of redox-sensitive core mitochondrial proteins. The g = 1.94 signal could prove to be an important biomarker for oxidative damage resulting from Cr(VI) exposure. The EPR spectra simultaneously showed signals for Cr(V) and Cr(III) which verify Cr(VI) exposure and its intracellular reductive activation.
doi:10.1016/j.freeradbiomed.2010.09.020
PMCID: PMC3005768  PMID: 20883776
chromate; chromium(VI); mitochondria; iron-sulfur proteins; aconitase; complex I; complex II
20.  Mitochondrial handling of excess Ca2+ is substrate-dependent with implications for reactive oxygen species generation 
Aim
The mitochondrial electron transport chain is the major source of reactive oxygen species (ROS) during cardiac ischemia. Several mechanisms modulate ROS production; one is mitochondrial Ca2+ uptake. Here we sought to elucidate the effects of extra-mitochondrial Ca2+ (e[Ca2+]) on ROS production (measured as H2O2 release) from complexes I and III.
Results
Mitochondria, isolated from guinea pig hearts, were pre-incubated with increasing concentrations of CaCl2 and then energized with the complex I substrate Na+-pyruvate or the complex II substrate Na+-succinate. Mitochondrial H2O2 release rates were assessed after giving either rotenone or antimycin A to inhibit complex I or III, respectively. After pyruvate, mitochondria maintained a fully polarized membrane potential (Δψ, assessed using rhodamine 123) and were able to generate NADH (assessed using autofluorescence) even with excess e[Ca2+] (assessed using CaGreen-5N), whereas they remained partially depolarized and did not generate NADH after succinate. This partial Δψ depolarization with succinate was accompanied by a large release of H2O2 (assessed using amplex red/horseradish peroxidase) with later addition of antimycin A. In the presence of excess e[Ca2+], adding cyclosporine A to inhibit mitochondrial permeability transition pore (mPTP) opening restored Δψ and significantly decreased antimycin A-induced H2O2 release.
Conclusions
Succinate accumulates during ischemia to become the major substrate utilized by cardiac mitochondria. The inability of mitochondria to maintain a fully polarized Δψ under excess e[Ca2+] when succinate, but not pyruvate, is the substrate may indicate a permeabilization of the mitochondrial membrane which enhances H2O2 emission from complex III during ischemia.
doi:10.1016/j.freeradbiomed.2012.09.020
PMCID: PMC3542420  PMID: 23010495
complex III; mitochondrial permeability transition pore; succinate; Ca2+
21.  Blockade of electron transport before ischemia protects mitochondria and decreases myocardial injury during reperfusion in aged rat hearts 
Myocardial injury is increased in the aged heart following ischemia and reperfusion (I-R) in both humans and experimental models. Hearts from aged 24 mo. old Fischer 344 rats sustain greater cell death and decreased contractile recovery after I-R compared to 6 mo. adult controls. Cardiac mitochondria incur damage during I-R contributing to cell death. Aged rats have a defect in complex III of the mitochondrial electron transport chain (ETC) localized to the interfibrillar population of cardiac mitochondria (IFM), situated in the interior of the cardiomyocyte among the myofibrils. The defect involves the quinol oxidation site (Qo) and increases the production of reactive oxygen species (ROS) in the baseline state. Ischemia further decreases complex III activity via functional inactivation of the iron-sulfur subunit. We studied the contribution of ischemia-induced defects in complex III to the increased cardiac injury in the aged heart. The reversible blockade of the ETC proximal to complex III during ischemia using amobarbital protects mitochondria against ischemic damage, removing the ischemia component of mitochondrial dysfunction. Reperfusion of the aged heart in the absence of ischemic mitochondrial damage decreases net ROS production from mitochondria and reduces cell death. Thus, even despite the persistence of the age-related defects in electron transport, protection against ischemic damage to mitochondria can reduce injury in the aged heart. The direct therapeutic targeting of mitochondria protects against ischemic damage and decreases cardiac injury during reperfusion in the high risk elderly heart.
doi:10.1016/j.trsl.2012.01.024
PMCID: PMC3423471  PMID: 22698829
ischemia; cytochrome c; myocardial infarction; aging; electron transport chain; reactive oxygen species
22.  Methamphetamine Causes Mitrochondrial Oxidative Damage in Human T Lymphocytes Leading to Functional Impairment 
Methamphetamine (METH) abuse is known to be associated with an inordinate rate of infections. Although many studies have described the association of METH exposure and immunosuppression, so far the underlying mechanism still remains elusive. In this study, we present evidence that METH exposure resulted in mitochondrial oxidative damage and caused dysfunction of primary human T cells. METH treatment of T lymphocytes led to a rise in intracellular calcium levels that enhanced the generation of reactive oxygen species. TCR-CD28 linked calcium mobilization and subsequent uptake by mitochondria in METH-treated T cells correlated with an increase in mitochondrion-derived superoxide. Exposure to METH-induced mitochondrial dysfunction in the form of marked decrease in mitochondrial membrane potential, increased mitochondrial mass, enhanced protein nitrosylation and diminished protein levels of complexes I, III, and IV of the electron transport chain. These changes paralleled reduced IL-2 secretion and T cell proliferative responses after TCR-CD28 stimulation indicating impaired T cell function. Furthermore, antioxidants attenuated METH-induced mitochondrial damage by preserving the protein levels of mitochondrial complexes I, III, and IV. Altogether, our data indicate that METH can cause T cell dysfunction via induction of oxidative stress and mitochondrial injury as underlying mechanism of immune impairment secondary to METH abuse.
doi:10.4049/jimmunol.0903691
PMCID: PMC3124898  PMID: 20668216
23.  Diffusive coupling can discriminate between similar reaction mechanisms in an allosteric enzyme system 
BMC Systems Biology  2010;4:165.
Background
A central question for the understanding of biological reaction networks is how a particular dynamic behavior, such as bistability or oscillations, is realized at the molecular level. So far this question has been mainly addressed in well-mixed reaction systems which are conveniently described by ordinary differential equations. However, much less is known about how molecular details of a reaction mechanism can affect the dynamics in diffusively coupled systems because the resulting partial differential equations are much more difficult to analyze.
Results
Motivated by recent experiments we compare two closely related mechanisms for the product activation of allosteric enzymes with respect to their ability to induce different types of reaction-diffusion waves and stationary Turing patterns. The analysis is facilitated by mapping each model to an associated complex Ginzburg-Landau equation. We show that a sequential activation mechanism, as implemented in the model of Monod, Wyman and Changeux (MWC), can generate inward rotating spiral waves which were recently observed as glycolytic activity waves in yeast extracts. In contrast, in the limiting case of a simple Hill activation, the formation of inward propagating waves is suppressed by a Turing instability. The occurrence of this unusual wave dynamics is not related to the magnitude of the enzyme cooperativity (as it is true for the occurrence of oscillations), but to the sensitivity with respect to changes of the activator concentration. Also, the MWC mechanism generates wave patterns that are more stable against long wave length perturbations.
Conclusions
This analysis demonstrates that amplitude equations, which describe the spatio-temporal dynamics near an instability, represent a valuable tool to investigate the molecular effects of reaction mechanisms on pattern formation in spatially extended systems. Using this approach we have shown that the occurrence of inward rotating spiral waves in glycolysis can be explained in terms of an MWC, but not with a Hill mechanism for the activation of the allosteric enzyme phosphofructokinase. Our results also highlight the importance of enzyme oligomerization for a possible experimental generation of Turing patterns in biological systems.
doi:10.1186/1752-0509-4-165
PMCID: PMC3014969  PMID: 21118520
24.  Cytoprotection by the Modulation of Mitochondrial Electron Transport Chain: The Emerging Role of Mitochondrial STAT3 
Mitochondrion  2011;12(2):180-189.
The down regulation of mitochondrial electron transport is an emerging mechanism of cytoprotective intervention that is effective in pathologic settings such as myocardial ischemia and reperfusion when the continuation of mitochondrial respiration produces reactive oxygen species, mitochondrial calcium overload, and the release of cytochrome c to activate cell death programs. The initial target of deranged electron transport is the mitochondria themselves. In the first part of this review, we describe this concept and summarize different approaches used to regulate mitochondrial respiration by targeting complex I as a proximal site in the electron transport chain (ETC) in order to favor the cytoprotection. The second part of the review highlights the emerging role of signal transducer and activator of transcription 3 (STAT3) in the direct, non-transcriptional regulation of ETC, as an example of a genetic approach to modulate respiration. Recent studies indicate that a pool of STAT3 resides in the mitochondria where it is necessary for the maximal activity of complexes I and II of the electron transport chain (ETC). The over expression of mitochondrial-targeted STAT3 results in a partial blockade of electron transport at complexes I and II that does not impair mitochondrial membrane potential nor enhance the production of reactive oxygen species (ROS). The targeting of transcriptionally-inactive STAT3 to mitochondria attenuates damage to mitochondria during cell stress, resulting in decreased production of ROS and retention of cytochrome c by mitochondria. The overexpression of STAT3 targeted to mitochondria unveils a novel protective approach mediated by modulation of mitochondrial respiration that is independent of STAT3 transcriptional activity. The limitation of mitochondrial respiration under pathologic circumstances can be approached by activation and over expression of endogenous signaling mechanisms in addition to pharmacologic means. The regulation of mitochondrial respiration comprises a cardioprotective paradigm to decrease cellular injury during ischemia and reperfusion.
doi:10.1016/j.mito.2011.08.011
PMCID: PMC3278553  PMID: 21930250
25.  Increased production of mitochondrial superoxide in the spinal cord induces pain behaviors in mice: The effect of mitochondrial electron transport complex inhibitors 
Neuroscience letters  2008;447(1):87-91.
Scavengers of reactive oxygen species (ROS) have been shown to produce a strong antinociceptive effect on persistent pain, and mitochondria are suggested to be the main source of ROS in the spinal dorsal horn. To explore whether excessive generation of mitochondrial superoxide alone can induce pain, the effect of mitochondrial electron transport complex inhibitors on the development of mechanical hyperalgesia was examined in mice. Intrathecal injection of an electron transport complex inhibitor, antimycin A or rotenone, in normal mice resulted in a slowly developing but long-lasting and dose-dependent mechanical hyperalgesia. The levels of mechanical hyperalgesia after antimycin A, a complex III inhibitor, were higher than that with rotenone, a complex I inhibitor. A large increase of mitochondrial superoxide in the spinal dorsal horn and a strong antinociceptive effect of ROS scavengers, phenyl-N-tert-butylnitrone (PBN) and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) were observed in antimycin A-treated mice. The study indicates that the enhanced production of spinal mitochondrial superoxide alone without nerve injury can produce mechanical hyperalgesia.
doi:10.1016/j.neulet.2008.09.041
PMCID: PMC2722070  PMID: 18832013
Mitochondrial ETC; Antimycin A; Rotenone; Mechanical hyperalgesia; ROS

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