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Reactive Oxygen Species Production by Mitochondria

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Mitochondrial DNA

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 554))

Abstract

Oxidative damage to cellular macromolecules is believed to underlie the development of many pathological states and aging. The agents responsible for this damage are generally thought to be reactive oxygen species, such as superoxide, hydrogen peroxide, and hydroxyl radical. The main source of reactive species production within most cells is the mitochondria. Within the mitochondria the primary reactive oxygen species produced is superoxide, most of which is converted to hydrogen peroxide by the action of superoxide dismutase. The production of superoxide by mitochondria has been localized to several enzymes of the electron transport chain, including Complexes I and III and glycerol-3-phosphate dehydrogenase. In this chapter the current consensus view of sites, rates, mechanisms, and topology of superoxide production by mitochondria is described. A brief overview of the methods for measuring reactive oxygen species production in isolated mitochondria and cells is also presented.

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References

  1. Harman, D. (1992) Free radical theory of aging. Mutat. Res. 275, 257–266.

    CAS  PubMed  Google Scholar 

  2. Droge, W. (2002) Free radicals in the physiological control of cell function. Physiol. Rev. 82, 47–95.

    CAS  PubMed  Google Scholar 

  3. McCord, J. M. and Fridovich, I. (1969) Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J. Biol. Chem. 244, 6049–6055.

    CAS  PubMed  Google Scholar 

  4. Lebovitz, R. M., Zhang, H., Vogel, H., Cartwright, J., Jr., Dionne, L., Lu, N., Huang, S. and Matzuk, M. M. (1996) Neurodegeneration, myocardial injury, and perinatal death in mitochondrial superoxide dismutase-deficient mice. Proc. Natl. Acad. Sci. USA 93, 9782–9787.

    Article  CAS  PubMed  Google Scholar 

  5. Li, Y., Huang, T. T., Carlson, E. J., Melov, S., Ursell, P. C., Olson, J. L., Noble, L. J., Yoshimura, M. P., Berger, C., Chan, P. H., Wallace, D. C. and Epstein, C. J. (1995) Dilated cardiomyopathy and neonatal lethality in mutant mice lacking manganese superoxide dismutase. Nat. Genet. 11, 376–381.

    Article  CAS  PubMed  Google Scholar 

  6. Halliwell, B. and Gutteridge, J. M. C. (1999) Free radicals in biology and medicine, Oxford University Press Inc, NY

    Google Scholar 

  7. Tarpey, M. M., Wink, D. A. and Grisham, M. B. (2004) Methods for detection of reactive metabolites of oxygen and nitrogen: in vitro and in vivo considerations. Am. J. Physiol. Regul. Integr. Comp. Physiol. 286, R431–444.

    CAS  Google Scholar 

  8. Gardner, P. R. (2002) Aconitase: sensitive target and measure of superoxide. Meth. Enzymol. 349, 9–23.

    Article  CAS  PubMed  Google Scholar 

  9. Talbot, D. A. and Brand, M. D. (2005) Uncoupling protein 3 protects aconitase against inactivation in isolated skeletal muscle mitochondria. Biochim. Biophys. Acta 1709, 150–156.

    CAS  PubMed  Google Scholar 

  10. Miwa, S. and Brand, M. D. (2005) The topology of superoxide production by complex III and glycerol 3-phosphate dehydrogenase in Drosophila mitochondria. Biochim. Biophys. Acta 1709, 214–219.

    Article  CAS  Google Scholar 

  11. St-Pierre, J., Buckingham, J. A., Roebuck, S. J. and Brand, M. D. (2002) Topology of superoxide production from different sites in the mitochondrial electron transport chain. J. Biol. Chem. 277, 44784–44790.

    Article  CAS  PubMed  Google Scholar 

  12. Benov, L., Sztejnberg, L. and Fridovich, I. (1998) Critical evaluation of the use of hydroethidine as a measure of superoxide anion radical. Free Radic. Biol. Med. 25, 826–831.

    Article  CAS  PubMed  Google Scholar 

  13. Budd, S. L., Castilho, R. F. and Nicholls, D. G. (1997) Mitochondrial membrane potential and hydroethidine-monitored superoxide generation in cultured cerebellar granule cells. FEBS Lett. 415, 21–24.

    Article  CAS  PubMed  Google Scholar 

  14. Zhao, H., Joseph, J., Fales, H. M., Sokoloski, E. A., Levine, R. L., Vasquez-Vivar, J. and Kalyanaraman, B. (2005) Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence. Proc. Natl. Acad. Sci. USA 102, 5727–5732.

    Article  CAS  PubMed  Google Scholar 

  15. Robinson, K. M., Janes, M. S., Pehar, M., Monette, J. S., Ross, M. F., Hagen, T. M., Murphy, M. P. and Beckman, J. S. (2006) Selective fluorescent imaging of superoxide in vivo using ethidium-based probes. Proc. Natl. Acad. Sci. USA 103, 15038–15043.

    Article  CAS  PubMed  Google Scholar 

  16. Negre-Salvayre, A., Auge, N., Duval, C., Robbesyn, F., Thiers, J. C., Nazzal, D., Benoist, H. and Salvayre, R (2002) Detection of intracellular reactive oxygen species in cultured cells using fluorescent probes. Meth. Enzymol. 352, 62–71.

    Article  CAS  PubMed  Google Scholar 

  17. O'Malley, Y. Q., Reszka, K. J. and Britigan, B. E. (2004) Direct oxidation of 2',7'-dichlorodihydrofluorescein by pyocyanin and other redox-active compounds independent of reactive oxygen species production. Free Radic. Biol. Med. 36, 90–100.

    Article  PubMed  Google Scholar 

  18. Rota, C., Chignell, C. F. and Mason, R. P. (1999) Evidence for free radical formation during the oxidation of 2'-7'- dichlorofluorescin to the fluorescent dye 2'-7'-dichlorofluorescein by horseradish peroxidase: possible implications for oxidative stress measurements. Free Radic. Biol. Med. 27, 873–881.

    Article  CAS  PubMed  Google Scholar 

  19. Hinkle, P. C., Butow, R. A., Racker, E. and Chance, B. (1967) Partial resolution of the enzymes catalyzing oxidative phosphorylation. XV. Reverse electron transfer in the flavin-cytochrome beta region of the respiratory chain of beef heart submitochondrial particles. J. Biol. Chem. 242, 5169–5173.

    CAS  PubMed  Google Scholar 

  20. Jensen, P. K. (1966) Antimycin-insensitive oxidation of succinate and reduced nicotinamide-adenine dinucleotide in electron-transport particles. I. pH dependency and hydrogen peroxide formation. Biochim. Biophys. Acta 122, 157–166.

    CAS  PubMed  Google Scholar 

  21. Loschen, G., Flohe, L. and Chance, B. (1971) Respiratory chain linked H2O2 production in pigeon heart mitochondria. FEBS Lett. 18, 261–264.

    Article  CAS  PubMed  Google Scholar 

  22. Andreyev, A. Y., Kushnareva, Y. E. and Starkov, A. A. (2005) Mitochondrial metabolism of reactive oxygen species. Biochemistry (Mosc) 70, 200–214.

    Article  CAS  Google Scholar 

  23. Brand, M. D., Affourtit, C., Esteves, T. C., Green, K., Lambert, A. J., Miwa, S., Pakay, J. L. and Parker, N. (2004) Mitochondrial superoxide: production, biological effects, and activation of uncoupling proteins. Free Radic. Biol. Med. 37, 755–767.

    Article  CAS  PubMed  Google Scholar 

  24. Jezek, P. and Hlavata, L. (2005) Mitochondria in homeostasis of reactive oxygen species in cell, tissues, and organism. Int. J. Biochem. Cell Biol. 37, 2478–2503.

    Article  CAS  PubMed  Google Scholar 

  25. Raha, S. and Robinson, B. H. (2000) Mitochondria, oxygen free radicals, disease and ageing. Trends Biochem. Sci. 25, 502–508.

    Article  CAS  PubMed  Google Scholar 

  26. Turrens, J. F. (2003) Mitochondrial formation of reactive oxygen species. J. Physiol. 552, 335–344.

    Article  CAS  PubMed  Google Scholar 

  27. Brandt, U. (2006) Energy converting NADH:quinone oxidoreductase (complex I). Annu. Rev. Biochem. 75, 69–92.

    Article  CAS  PubMed  Google Scholar 

  28. Hansford, R. G., Hogue, B. A. and Mildaziene, V. (1997) Dependence of H2O2 formation by rat heart mitochondria on substrate availability and donor age. J. Bioenerg. Biomembr. 29, 89–95.

    Article  CAS  PubMed  Google Scholar 

  29. Kushnareva, Y., Murphy, A. N. and Andreyev, A. (2002) Complex I-mediated reactive oxygen species generation: modulation by cytochrome c and NAD(P)+ oxidation-reduction state. Biochem. J. 368, 545–553.

    Article  CAS  PubMed  Google Scholar 

  30. Lambert, A. J. and Brand, M. D. (2004) Superoxide production by NADH:ubiquinone oxidoreductase (complex I) depends on the pH gradient across the mitochondrial inner membrane. Biochem. J. 382, 511–517.

    Article  CAS  PubMed  Google Scholar 

  31. Liu, Y., Fiskum, G. and Schubert, D. (2002) Generation of reactive oxygen species by the mitochondrial electron transport chain. J. Neurochem. 80, 780–787.

    Article  CAS  PubMed  Google Scholar 

  32. Votyakova, T. V. and Reynolds, I. J. (2001) ΔΨm-Dependent and -independent production of reactive oxygen species by rat brain mitochondria. J. Neurochem. 79, 266–277.

    Article  CAS  PubMed  Google Scholar 

  33. Ohnishi, S. T., Ohnishi, T., Muranaka, S., Fujita, H., Kimura, H., Uemura, K., Yoshida, K. and Utsumi, K. (2005) A possible site of superoxide generation in the complex I segment of rat heart mitochondria. J. Bioenerg. Biomembr. 37, 1–15.

    Article  CAS  PubMed  Google Scholar 

  34. Gyulkhandanyan, A. V. and Pennefather, P. S. (2004) Shift in the localization of sites of hydrogen peroxide production in brain mitochondria by mitochondrial stress. J. Neurochem. 90, 405–421.

    Article  CAS  PubMed  Google Scholar 

  35. Miwa, S., St-Pierre, J., Partridge, L. and Brand, M. D. (2003) Superoxide and hydrogen peroxide production by Drosophila mitochondria. Free Radic. Biol. Med. 35, 938–948.

    Article  CAS  Google Scholar 

  36. Kwong, L. K. and Sohal, R. S. (1998) Substrate and site specificity of hydrogen peroxide generation in mouse mitochondria. Arch. Biochem. Biophys. 350, 118–126.

    Article  CAS  PubMed  Google Scholar 

  37. Lambert, A. J., Boysen, H. M., Buckingham, J. A., Yang, T., Podlutsky, A., Austad, S. N., Kunz, T. H., Buffenstein, R. and Brand, M. D. (2007) Low rates of hydrogen peroxide production by isolated heart mitochondria associate with long maximum lifespan in vertebrate homeotherms. Aging Cell 6, 607–618.

    Google Scholar 

  38. Li, Y. and Trush, M. A. (1998) Diphenyleneiodonium, an NAD(P)H oxidase inhibitor, also potently inhibits mitochondrial reactive oxygen species production. Biochem. Biophys. Res. Commun. 253, 295–299.

    Article  CAS  PubMed  Google Scholar 

  39. Parthasarathi, K., Ichimura, H., Quadri, S., Issekutz, A. and Bhattacharya, J. (2002) Mitochondrial reactive oxygen species regulate spatial profile of proinflammatory responses in lung venular capillaries. J. Immunol. 169, 7078–7086.

    CAS  PubMed  Google Scholar 

  40. Schuchmann, S. and Heinemann, U. (2000) Increased mitochondrial superoxide generation in neurons from trisomy 16 mice: a model of Down's syndrome. Free Radic. Biol. Med. 28, 235–250.

    Article  CAS  PubMed  Google Scholar 

  41. Vrablic, A. S., Albright, C. D., Craciunescu, C. N., Salganik, R. I. and Zeisel, S. H. (2001) Altered mitochondrial function and overgeneration of reactive oxygen species precede the induction of apoptosis by 1-O-octadecyl-2-methyl-rac-glycero-3-phosphocholine in p53-defective hepatocytes. FASEB J. 15, 1739–1744.

    Article  CAS  PubMed  Google Scholar 

  42. Barrientos, A. and Moraes, C. T. (1999) Titrating the effects of mitochondrial complex I impairment in the cell physiology. J. Biol. Chem. 274, 16188–16197.

    Article  CAS  PubMed  Google Scholar 

  43. Li, N., Ragheb, K., Lawler, G., Sturgis, J., Rajwa, B., Melendez, J. A. and Robinson, J. P. (2003) Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production. J. Biol. Chem. 278, 8516–8525.

    Article  CAS  PubMed  Google Scholar 

  44. Nakamura, K., Bindokas, V. P., Kowlessur, D., Elas, M., Milstien, S., Marks, J. D., Halpern, H. J. and Kang, U. J. (2001) Tetrahydrobiopterin scavenges superoxide in dopaminergic neurons. J. Biol. Chem. 276, 34402–34407.

    Article  CAS  PubMed  Google Scholar 

  45. Siraki, A. G., Pourahmad, J., Chan, T. S., Khan, S. and O'Brien, P. J. (2002) Endogenous and endobiotic induced reactive oxygen species formation by isolated hepatocytes. Free Radic. Biol. Med. 32, 2–10.

    Article  CAS  PubMed  Google Scholar 

  46. Kudin, A. P., Bimpong-Buta, N. Y., Vielhaber, S., Elger, C. E. and Kunz, W. S. (2004) Characterization of superoxide-producing sites in isolated brain mitochondria. J. Biol. Chem. 279, 4127–4135.

    Article  CAS  PubMed  Google Scholar 

  47. Lambert, A. J. and Brand, M. D. (2004) Inhibitors of the quinone-binding site allow rapid superoxide production from mitochondrial NADH:ubiquinone oxidoreductase (Complex I). J. Biol. Chem. 279, 39414–39420.

    Article  CAS  PubMed  Google Scholar 

  48. Barja, G. and Herrero, A. (1998) Localization at complex I and mechanism of the higher free radical production of brain nonsynaptic mitochondria in the short-lived rat than in the longevous pigeon. J. Bioenerg. Biomembr. 30, 235–243.

    Article  CAS  PubMed  Google Scholar 

  49. Herrero, A. and Barja, G. (1997) Sites and mechanisms responsible for the low rate of free radical production of heart mitochondria in the long-lived pigeon. Mech. Ageing Dev. 98, 95–111.

    Article  CAS  PubMed  Google Scholar 

  50. Herrero, A. and Barja, G. (1998) H2O2 production of heart mitochondria and aging rate are slower in canaries and parakeets than in mice: sites of free radical generation and mechanisms involved. Mech. Ageing Dev. 103, 133–146.

    Article  CAS  PubMed  Google Scholar 

  51. Korshunov, S. S., Skulachev, V. P. and Starkov, A. A. (1997) High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Lett. 416, 15–18.

    Article  CAS  PubMed  Google Scholar 

  52. Liu, S. S. (1997) Generating, partitioning, targeting and functioning of superoxide in mitochondria. Biosci. Rep. 17, 259–272.

    Article  CAS  PubMed  Google Scholar 

  53. Brand, M. D. (2000) Uncoupling to survive? The role of mitochondrial inefficiency in ageing. Exp. Gerontol. 35, 811–820.

    Article  CAS  PubMed  Google Scholar 

  54. Speakman, J. R., Talbot, D. A., Selman, C., Snart, S., McLaren, J. S., Redman, P., Krol, E., Jackson, D. M., Johnson, M. S. and Brand, M. D. (2004) Uncoupled and surviving: individual mice with high metabolism have greater mitochondrial uncoupling and live longer. Aging Cell 3, 87–95.

    Article  CAS  PubMed  Google Scholar 

  55. Kussmaul, L. and Hirst, J. (2006) The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria. Proc. Natl. Acad. Sci. USA 103, 7607–7612.

    Article  CAS  PubMed  Google Scholar 

  56. Johnson, J. E., Jr., Choksi, K. and Widger, W. R. (2003) NADH-Ubiquinone oxidoreductase: substrate-dependent oxygen turnover to superoxide anion as a function of flavin mononucleotide. Mitochondrion 3, 97–110.

    Article  PubMed  Google Scholar 

  57. Genova, M. L., Ventura, B., Giuliano, G., Bovina, C., Formiggini, G., Parenti Castelli, G. and Lenaz, G. (2001) The site of production of superoxide radical in mitochondrial complex I is not a bound ubisemiquinone but presumably iron-sulfur cluster N2. FEBS Lett. 505, 364–368.

    Article  CAS  PubMed  Google Scholar 

  58. Herrero, A. and Barja, G. (2000) Localization of the site of oxygen radical generation inside the complex I of heart and nonsynaptic brain mammalian mitochondria. J. Bioenerg. Biomembr. 32, 609–615.

    Article  CAS  PubMed  Google Scholar 

  59. Muller, F. L., Liu, Y. and Van Remmen, H. (2004) Complex III releases superoxide to both sides of the inner mitochondrial membrane. J. Biol. Chem. 279, 49064–49073.

    Article  CAS  PubMed  Google Scholar 

  60. Trumpower, B. L. (1990) The protonmotive Q cycle. Energy transduction by coupling of proton translocation to electron transfer by the cytochrome bc 1 complex. J. Biol. Chem. 265, 11409–11412.

    CAS  PubMed  Google Scholar 

  61. Chen, Q., Vazquez, E. J., Moghaddas, S., Hoppel, C. L. and Lesnefsky, E. J. (2003) Production of reactive oxygen species by mitochondria: central role of complex III. J. Biol. Chem. 278, 36027–36031.

    Article  CAS  PubMed  Google Scholar 

  62. McLennan, H. R. and Degli Esposti, M. (2000) The contribution of mitochondrial respiratory complexes to the production of reactive oxygen species. J. Bioenerg. Biomembr. 32, 153–162.

    Article  CAS  PubMed  Google Scholar 

  63. Raha, S., McEachern, G. E., Myint, A. T. and Robinson, B. H. (2000) Superoxides from mitochondrial complex III: the role of manganese superoxide dismutase. Free Radic. Biol. Med. 29, 170–180.

    Article  CAS  PubMed  Google Scholar 

  64. Cape, J. L., Bowman, M. K. and Kramer, D. M. (2007) A semiquinone intermediate generated at the Qo site of the cytochrome bc1 complex: Importance for the Q-cycle and superoxide production. Proc. Natl. Acad. Sci. USA 104, 7887–7892.

    Article  CAS  PubMed  Google Scholar 

  65. Turrens, J. F., Alexandre, A. and Lehninger, A. L. (1985) Ubisemiquinone is the electron donor for superoxide formation by complex III of heart mitochondria. Arch. Biochem. Biophys. 237, 408–414.

    Article  CAS  PubMed  Google Scholar 

  66. Talbot, D. A., Lambert, A. J. and Brand, M. D. (2004) Production of endogenous matrix superoxide from mitochondrial complex I leads to activation of uncoupling protein 3. FEBS Lett. 556, 111–115.

    Article  CAS  PubMed  Google Scholar 

  67. Han, D., Williams, E. and Cadenas, E. (2001) Mitochondrial respiratory chain-dependent generation of superoxide anion and its release into the intermembrane space. Biochem. J. 353, 411–416.

    Article  CAS  PubMed  Google Scholar 

  68. Rustin, P., Munnich, A. and Rotig, A. (2002) Succinate dehydrogenase and human diseases: new insights into a well-known enzyme. Eur. J. Hum. Genet. 10, 289–291.

    Article  CAS  PubMed  Google Scholar 

  69. Senoo-Matsuda, N., Yasuda, K., Tsuda, M., Ohkubo, T., Yoshimura, S., Nakazawa, H., Hartman, P. S. and Ishii, N. (2001) A defect in the cytochrome b large subunit in complex II causes both superoxide anion overproduction and abnormal energy metabolism in Caenorhabditis elegans. J. Biol. Chem. 276, 41553–41558.

    Article  CAS  PubMed  Google Scholar 

  70. Messner, K. R. and Imlay, J. A. (2002) Mechanism of superoxide and hydrogen peroxide formation by fumarate reductase, succinate dehydrogenase, and aspartate oxidase. J. Biol. Chem. 277, 42563–42571.

    Article  CAS  PubMed  Google Scholar 

  71. Tretter, L., Takacs, K., Hegedus, V. and Adam-Vizi, V. (2007) Characteristics of alpha-glycerophosphate-evoked H2O2 generation in brain mitochondria. J. Neurochem. 100, 650–663.

    Article  CAS  PubMed  Google Scholar 

  72. Drahota, Z., Chowdhury, S. K., Floryk, D., Mracek, T., Wilhelm, J., Rauchova, H., Lenaz, G. and Houstek, J. (2002) Glycerophosphate-dependent hydrogen peroxide production by brown adipose tissue mitochondria and its activation by ferricyanide. J. Bioenerg. Biomembr. 34, 105–113.

    Article  CAS  PubMed  Google Scholar 

  73. Sekhar, B. S., Kurup, C. K. and Ramasarma, T. (1987) Generation of hydrogen peroxide by brown adipose tissue mitochondria. J. Bioenerg. Biomembr. 19, 397–407.

    Article  CAS  PubMed  Google Scholar 

  74. Gazaryan, I. G., Krasnikov, B. F., Ashby, G. A., Thorneley, R. N., Kristal, B. S. and Brown, A. M. (2002) Zinc is a potent inhibitor of thiol oxidoreductase activity and stimulates reactive oxygen species production by lipoamide dehydrogenase. J. Biol. Chem. 277, 10064–10072.

    Article  CAS  PubMed  Google Scholar 

  75. Starkov, A. A., Fiskum, G., Chinopoulos, C., Lorenzo, B. J., Browne, S. E., Patel, M. S. and Beal, M. F. (2004) Mitochondrial alpha-ketoglutarate dehydrogenase complex generates reactive oxygen species. J. Neurosci. 24, 7779–7788.

    Article  CAS  PubMed  Google Scholar 

  76. Fang, J. and Beattie, D.S. (2003) External alternative NADH dehydrogenase of Saccharomyces cerevisiae: a potential source of superoxide. Free Radic. Biol. Med. 34, 478–488.

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. MRC Dunn Human Nutrition Unit, Cambridge, UK

    Adrian J. Lambert

  2. Buck Institute of Aging, Novato, CA, USA

    Martin D. Brand

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  1. Adrian J. Lambert
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  1. Department of Biological Sciences, Brock University, 500 Glenridge Avenue, Cpy Catharines, ON, L2S 3A1, Canada

    Jeffrey A. Stuart

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Lambert, A.J., Brand, M.D. (2009). Reactive Oxygen Species Production by Mitochondria. In: Stuart, J.A. (eds) Mitochondrial DNA. Methods in Molecular Biology™, vol 554. Humana Press. https://doi.org/10.1007/978-1-59745-521-3_11

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  • DOI: https://doi.org/10.1007/978-1-59745-521-3_11

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-934115-60-2

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