Complex I-mediated reactive oxygen species generation: modulation by cytochrome c and NAD(P)+ oxidation–reduction state

Several lines of evidence indicate that mitochondrial reactive oxygen species (ROS) generation is the major source of oxidative stress in the cell. It has been shown that ROS production accompanies cytochrome c release in different apoptotic paradigms, but the site(s) of ROS production remain obscure. In the current study, we demonstrate that loss of cytochrome c by mitochondria oxidizing NAD+-linked substrates results in a dramatic increase of ROS production and respiratory inhibition. This increased ROS production can be mimicked by rotenone, a complex I inhibitor, as well as other chemical inhibitors of electron flow that act further downstream in the electron transport chain. The effects of cytochrome c depletion from mitoplasts on ROS production and respiration are reversible upon addition of exogenous cytochrome c. Thus in these models of mitochondrial injury, a primary site of ROS generation in both brain and heart mitochondria is proximal to the rotenone inhibitory site, rather than in complex III. ROS production at complex I is critically dependent upon a highly reduced state of the mitochondrial NAD(P)+ pool and is achieved upon nearly complete inhibition of the respiratory chain. Redox clamp experiments using the acetoacetate/d-β-hydroxybutyrate couple in the presence of a maximally inhibitory rotenone concentration suggest that the site is approx. 50mV more electronegative than the NADH/NAD+ couple. In the absence of inhibitors, this highly reduced state of mitochondria can be induced by reverse electron flow from succinate to NAD+, accounting for profound ROS production in the presence of succinate. These results lead us to propose a model of thermodynamic control of mitochondrial ROS production which suggests that the ROS-generating site of complex I is the Fe—S centre N-1a.

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Oxygen Sensitivity of Mitochondrial Reactive Oxygen Species Generation Depends on Metabolic Conditions

Journal of Biological Chemistry ◽

10.1074/jbc.m809512200 ◽

2009 ◽

Vol 284 (24) ◽

pp. 16236-16245 ◽

Cited By ~ 116

Author(s):

David L. Hoffman ◽

Paul S. Brookes

Keyword(s):

Reactive Oxygen Species ◽

Electron Transfer ◽

Complex I ◽

Reactive Oxygen ◽

Complex Iii ◽

Ros Generation ◽

Oxygen Species ◽

Quinone Oxidoreductase ◽

Electron Transfer Flavoprotein ◽

Metabolic Conditions

The mitochondrial generation of reactive oxygen species (ROS) plays a central role in many cell signaling pathways, but debate still surrounds its regulation by factors, such as substrate availability, [O2] and metabolic state. Previously, we showed that in isolated mitochondria respiring on succinate, ROS generation was a hyperbolic function of [O2]. In the current study, we used a wide variety of substrates and inhibitors to probe the O2 sensitivity of mitochondrial ROS generation under different metabolic conditions. From such data, the apparent Km for O2 of putative ROS-generating sites within mitochondria was estimated as follows: 0.2, 0.9, 2.0, and 5.0 μm O2 for the complex I flavin site, complex I electron backflow, complex III QO site, and electron transfer flavoprotein quinone oxidoreductase of β-oxidation, respectively. Differential effects of respiratory inhibitors on ROS generation were also observed at varying [O2]. Based on these data, we hypothesize that at physiological [O2], complex I is a significant source of ROS, whereas the electron transfer flavoprotein quinone oxidoreductase may only contribute to ROS generation at very high [O2]. Furthermore, we suggest that previous discrepancies in the assignment of effects of inhibitors on ROS may be due to differences in experimental [O2]. Finally, the data set (see supplemental material) may be useful in the mathematical modeling of mitochondrial metabolism.

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Caspase-mediated loss of mitochondrial function and generation of reactive oxygen species during apoptosis

The Journal of Cell Biology ◽

10.1083/jcb.200208089 ◽

2003 ◽

Vol 160 (1) ◽

pp. 65-75 ◽

Cited By ~ 338

Author(s):

Jean-Ehrland Ricci ◽

Roberta A. Gottlieb ◽

Douglas R. Green

Keyword(s):

Reactive Oxygen Species ◽

Oxygen Consumption ◽

Electron Transport ◽

Cytochrome C ◽

Mitochondrial Function ◽

Complex I ◽

Caspase 3 ◽

Reactive Oxygen ◽

Complex Iii ◽

Oxygen Species

During apoptosis, the permeabilization of the mitochondrial outer membrane allows the release of cytochrome c, which induces caspase activation to orchestrate the death of the cell. Mitochondria rapidly lose their transmembrane potential (ΔΨm) and generate reactive oxygen species (ROS), both of which are likely to contribute to the dismantling of the cell. Here we show that both the rapid loss of ΔΨm and the generation of ROS are due to the effects of activated caspases on mitochondrial electron transport complexes I and II. Caspase-3 disrupts oxygen consumption induced by complex I and II substrates but not that induced by electron transfer to complex IV. Similarly, ΔΨm generated in the presence of complex I or II substrates is disrupted by caspase-3, and ROS are produced. Complex III activity measured by cytochrome c reduction remains intact after caspase-3 treatment. In apoptotic cells, electron transport and oxygen consumption that depends on complex I or II was disrupted in a caspase-dependent manner. Our results indicate that after cytochrome c release the activation of caspases feeds back on the permeabilized mitochondria to damage mitochondrial function (loss of ΔΨm) and generate ROS through effects of caspases on complex I and II in the electron transport chain.

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Reactive Oxygen Species in Host Plant Are Required for an Early Defense Response against Attack of Stagonospora nodorum Berk. Necrotrophic Effectors SnTox

Plants ◽

10.3390/plants10081586 ◽

2021 ◽

Vol 10 (8) ◽

pp. 1586

Author(s):

Svetlana Veselova ◽

Tatyana Nuzhnaya ◽

Guzel Burkhanova ◽

Sergey Rumyantsev ◽

Igor Maksimov

Keyword(s):

Reactive Oxygen Species ◽

Early Stage ◽

Reactive Oxygen ◽

Triticum Aestivum L ◽

Redox Status ◽

Stagonospora Nodorum ◽

Ros Generation ◽

Ros Production ◽

Oxygen Species ◽

Necrotrophic Effectors

Reactive oxygen species (ROS) play a central role in plant immune responses. The most important virulence factors of the Stagonospora nodorum Berk. are multiple fungal necrotrophic effectors (NEs) (SnTox) that affect the redox-status and cause necrosis and/or chlorosis in wheat lines possessing dominant susceptibility genes (Snn). However, the effect of NEs on ROS generation at the early stages of infection has not been studied. We studied the early stage of infection of various wheat genotypes with S nodorum isolates -Sn4VD, SnB, and Sn9MN, carrying a different set of NE genes. Our results indicate that all three NEs of SnToxA, SnTox1, SnTox3 significantly contributed to cause disease, and the virulence of the isolates depended on their differential expression in plants (Triticum aestivum L.). The Tsn1–SnToxA, Snn1–SnTox1and Snn3–SnTox3 interactions played an important role in inhibition ROS production at the initial stage of infection. The Snn3–SnTox3 inhibited ROS production in wheat by affecting NADPH-oxidases, peroxidases, superoxide dismutase and catalase. The Tsn1–SnToxA inhibited ROS production in wheat by affecting peroxidases and catalase. The Snn1–SnTox1 inhibited the production of ROS in wheat by mainly affecting a peroxidase. Collectively, these results show that the inverse gene-for gene interactions between effector of pathogen and product of host sensitivity gene suppress the host’s own PAMP-triggered immunity pathway, resulting in NE-triggered susceptibility (NETS). These results are fundamentally changing our understanding of the development of this economical important wheat disease.

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Abstract 19967: Differential Roles of the NADPH-Oxidase 1 and 2 in Platelet Activation and Thrombosis

Circulation ◽

10.1161/circ.132.suppl_3.19967 ◽

2015 ◽

Vol 132 (suppl_3) ◽

Author(s):

Michael K Delaney ◽

Kyungho Kim ◽

Brian Estevez ◽

Aleksandra Stojanovic-Terpo ◽

Bo Shen ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Platelet Activation ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Reactive Oxygen ◽

Ros Generation ◽

Ros Production ◽

Oxygen Species ◽

General Role ◽

Glycoprotein Vi ◽

Activation Pathways

Objective: Reactive oxygen species (ROS) generated from activated platelets is known to regulate platelet activation. However, it remains unclear whether and how different isoforms of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases (NOXs) play roles in different platelet activation pathways. Here we investigated the role of NOX1 and NOX2 in different platelet activation pathways using NOX1 and NOX2 knockout mice. Approach and Results: NOX1-/- platelets showed selective defects in G protein coupled receptor (GPCR)-mediated platelet activation induced by thrombin, protease-activated receptor 4 agonist peptide (PAR4AP) and thromboxane A2 analog U46619, but was not affected in platelet activation induced by collagen-related peptide (CRP), a glycoprotein VI (GPVI) agonist. In contrast, NOX2-/- platelets showed potent inhibition of CRP-induced platelet activation, and also showed partial inhibition of thrombin-induced platelet aggregation and secretion. Consistently, production of reactive oxygen species (ROS) was inhibited in NOX1-/- platelets stimulated with thrombin, but not CRP, whereas NOX2-/- platelets showed reduced ROS generation induced by CRP or thrombin. Interestingly, laser-induced arterial thrombosis was impaired in NOX2-/- mice, and in thrombocytopenic mice transfused with NOX2-/- platelets, suggesting an important role for NOX2-dependent platelet ROS production in the laser-induced injury model of thrombosis. Conclusions: NOX1 and NOX2 play differential roles in different platelet activation pathways: NOX1 mediates GPCR-mediated ROS production and platelet activation, whereas NOX2 plays a general role in GPVI- and GPCR-induced ROS production and platelet activation in vitro , and in laser-induced thrombosis in vivo .

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Isoflurane Differentially Modulates Mitochondrial Reactive Oxygen Species Production via Forward versus Reverse Electron Transport Flow

Anesthesiology ◽

10.1097/aln.0b013e31822a2316 ◽

2011 ◽

Vol 115 (3) ◽

pp. 531-540 ◽

Cited By ~ 51

Author(s):

Naoyuki Hirata ◽

Yon Hee Shim ◽

Danijel Pravdic ◽

Nicole L. Lohr ◽

Philip F. Pratt ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Electron Transport ◽

Complex I ◽

Ros Generation ◽

Ros Production ◽

Oxygen Species ◽

Submitochondrial Particles ◽

Anesthetic Preconditioning ◽

Cardioprotective Effects ◽

Complex I Activity

Background Reactive oxygen species (ROS) mediate the effects of anesthetic precondition to protect against ischemia and reperfusion injury, but the mechanisms of ROS generation remain unclear. In this study, the authors investigated if mitochondria-targeted antioxidant (mitotempol) abolishes the cardioprotective effects of anesthetic preconditioning. Further, the authors investigated the mechanism by which isoflurane alters ROS generation in isolated mitochondria and submitochondrial particles. Methods Rats were pretreated with 0.9% saline, 3.0 mg/kg mitotempol in the absence or presence of 30 min exposure to isoflurane. Myocardial infarction was induced by left anterior descending artery occlusion for 30 min followed by reperfusion for 2 h and infarct size measurements. Mitochondrial ROS production was determined spectrofluorometrically. The effect of isoflurane on enzymatic activity of mitochondrial respiratory complexes was also determined. Results Isoflurane reduced myocardial infarct size (40 ± 9% = mean ± SD) compared with control experiments (60 ± 4%). Mitotempol abolished the cardioprotective effects of anesthetic preconditioning (60 ± 9%). Isoflurane enhanced ROS generation in submitochondrial particles with nicotinamide adenine dinucleotide (reduced form), but not with succinate, as substrate. In intact mitochondria, isoflurane enhanced ROS production in the presence of rotenone, antimycin A, or ubiquinone when pyruvate and malate were substrates, but isoflurane attenuated ROS production when succinate was substrate. Mitochondrial respiratory experiments and electron transport chain complex assays revealed that isoflurane inhibited only complex I activity. Conclusions The results demonstrated that isoflurane produces ROS at complex I and III of the respiratory chain via the attenuation of complex I activity. The action on complex I decreases unfavorable reverse electron flow and ROS release in myocardium during reperfusion.

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Impact of CD14 on Reactive Oxygen Species Production from Human Leukocytes Primed byEscherichia coliLipopolysaccharides

Oxidative Medicine and Cellular Longevity ◽

10.1155/2019/6043245 ◽

2019 ◽

Vol 2019 ◽

pp. 1-9 ◽

Cited By ~ 1

Author(s):

Dmitry S. Kabanov ◽

Olga Yu. Vwedenskaya ◽

Marina A. Fokina ◽

Elena M. Morozova ◽

Sergey V. Grachev ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Reactive Oxygen ◽

Polymorphonuclear Neutrophils ◽

Ros Generation ◽

Ros Production ◽

Oxygen Species ◽

Human Pmns ◽

Species Production ◽

The Impact ◽

Priming Activity

Lipopolysaccharides (LPS) from Gram-negative bacteria prime human polymorphonuclear neutrophils (PMNs) via multicomponent receptor cluster including CD14 and MD-2·TLR4 for the enhanced release of reactive oxygen species (ROS) were triggered by bacterial derived peptideN-formyl-methionyl-leucyl-phenylalanine (fMLP). In this study, we investigated the impact of CD14 on LPS-induced priming of human PMNs for fMLP-triggered ROS generation (respiratory or oxidative) burst. Monoclonal antibodies against human CD14 (mAbs) as well as isotype-matched IgG2a did not influence significantly fMLP-triggered ROS production from LPS-unprimed PMNs. Anti-CD14 mAbs (clone UCHM-1) attenuated LPS-induced priming of PMNs as it had been mirrored by fMLP-triggered decrease of ROS production. Similar priming activity of S-LPS or Re-LPS fromEscherichia colifor fMLP-triggered ROS release from PMNs was found. Obtained results suggest that glycosylphosphatidylinositol-anchored CD14 is the key player in LPS-induced PMN priming for fMLP-triggered ROS production. We believe that blockade of CD14 on the cell surface and clinical use of anti-CD14 mAbs or their Fab fragments may diminish the production of ROS and improve outcomes during cardiovascular diseases manifested by LPS-induced inflammation.

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Reactive Oxygen Species Are Differentially Regulated in Chronic Myeloid Leukemia Versus Philadelphia Negative Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v112.11.4215.4215 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4215-4215

Author(s):

Estelle Guerin ◽

Francis Belloc ◽

Gabriel Etienne ◽

Pierre Duffau ◽

Francois-Xavier Mahon ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Bone Marrow ◽

Peripheral Blood ◽

Myeloproliferative Neoplasms ◽

Reactive Oxygen ◽

Ros Generation ◽

Separate Analysis ◽

Ros Production ◽

Oxygen Species ◽

Normal Cells

Abstract Deregulation of tyrosine-kinases is a characteristic of most Myeloproliferative Neoplasms (MPN); evolution from chronic phase to acute leukemia depends on the acquisition of additional mutations. Reactive Oxygen Species (ROS), the production of which is increased by tyrosine-kinase activation, can be responsible for additional mutations. The role of ROS in generating genetic aberrations has been mainly studied in BCR-ABL-positive cell lines. Little is known of ROS metabolism in primary cells from CML or Philadelphia-negative MPN (Ph-MPN). After informed consent, cells from blood or bone marrow were obtained from patients diagnosed with CML (12 bone marrow (BM), 8 peripheral blood (PB)), or Ph-MPN (4 Polycythemia Vera, 6 Essential Thrombocythemia, 3 Primary Myelofibroses, 2 atypical CML) and from healthy donors (bone marrow donors) or patients devoid of hematological disease undergoing thoracotomy. Cells were incubated with DCFDA, a fluorogenic marker of ROS production, labelled with an anti-CD45 antibody, stimulated with either the oxidant hydrogen peroxide (H2O2) or the PKC activator Phorbol Myristate Acetate (PMA), and analysed for ROS production by flow cytometry. CD45/SSC gating allowed separate analysis of granulocytes, monocytes or lymphocytes. The basal level of ROS was not higher in CML cells as compared to normal BM or PB leukocytes. It was even significantly lower in CML lymphocytes, either from the BM (2.35 Arbitrary Units vs 8.3 AU, p=5.5 10−5) or PB (2.47 AU vs 7.4 AU, p=3.10−5) and in CML granulocytes from peripheral blood (14 AU vs 45 AU, p =10 −5), but not bone marrow. The ROS levels of Ph-MPN cells were similar or slightly higher than control cells. Upon H2O2 stimulation however, ROS production increased significantly more in CML cells as compared to normal cells (6 fold increase), whatever the cell type (granulocytes, monocytes and lymphocytes) or their origin (PB or BM). In contrast, for Ph-MPN cells, H2O2-stimulated ROS production was close to that of normal cells, with only BM lymphocytes showing ROS generation four fold higher than control BM lymphocytes. After PMA stimulation, which yielded a more modest ROS production than H2O2, CML cells behaved similarly to normal cells, whereas ROS production was four fold higher in Ph-MPN cells, whatever their type and origin. In conclusion, ROS levels at the basal stage are not higher in MPN cells, whether they are Philadelphia positive or negative, as compared to normal cells. Various kinds of stimulation induce different patterns of response, CML cells being more sensitive to oxidants whereas Ph-MPN cells respond more to the cytokine-mimicking agent PMA. These results suggest that the mechanisms of ROS generation and thus of genetic instability are different in CML and Ph-MPN.

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Electron flow into cytochrome c coupled with reactive oxygen species from the electron transport chain converts cytochrome c to a cardiolipin peroxidase: role during ischemia–reperfusion

Biochimica et Biophysica Acta (BBA) - General Subjects ◽

10.1016/j.bbagen.2014.07.017 ◽

2014 ◽

Vol 1840 (11) ◽

pp. 3199-3207 ◽

Cited By ~ 24

Author(s):

Hema S. Aluri ◽

David C. Simpson ◽

Jeremy C. Allegood ◽

Ying Hu ◽

Karol Szczepanek ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Electron Transport ◽

Cytochrome C ◽

Electron Transport Chain ◽

Ischemia Reperfusion ◽

Electron Flow ◽

Reactive Oxygen ◽

Oxygen Species ◽

Transport Chain

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Bufalin Induces Reactive Oxygen Species Dependent Bax Translocation and Apoptosis in ASTC-a-1 Cells

Evidence-based Complementary and Alternative Medicine ◽

10.1093/ecam/nep082 ◽

2011 ◽

Vol 2011 ◽

pp. 1-12 ◽

Cited By ~ 21

Author(s):

Lei Sun ◽

Tongsheng Chen ◽

Xiaoping Wang ◽

Yun Chen ◽

Xunbin Wei

Keyword(s):

Reactive Oxygen Species ◽

Cell Death ◽

Molecular Mechanisms ◽

Caspase 3 ◽

Temporal Dynamics ◽

Reactive Oxygen ◽

Ros Generation ◽

Ros Production ◽

Oxygen Species ◽

Induced Apoptosis

Bufalin has been shown to induce cancer cell death through apoptotic pathways. However, the molecular mechanisms are not well understood. In this study, we used the confocal fluorescence microscopy (CFM) to monitor the spatio-temporal dynamics of reactive oxygen species (ROS) production, Bax translocation and caspase-3 activation during bufalin-induced apoptosis in living human lung adenocarcinoma (ASTC-a-1) cells. Bufalin induced ROS production and apoptotic cell death, demonstrated by Hoechst 33258 staining as well as flow cytometry analysis. Bax redistributed from cytosol to mitochondria from 12 to 48 h after bufalin treatment in living cells expressed with green fluorescent protein Bax. Treatment with the antioxidantN-acetyl-cysteine (NAC), a ROS scavenger, inhibited ROS generation and Bax translocation and led to a significant protection against bufalin-induced apoptosis. Our results also revealed that bufalin induced a prominent increase of caspase-3 activation blocked potently by NAC. Taken together, bufalin induced ROS-mediated Bax translocation, mitochondrial permeability transition and caspase-3 activation, implying that bufalin induced apoptosis via ROS-dependent mitochondrial death pathway in ASTC-a-1 cells.

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