The oxidative inactivation of mitochondrial electron transport chain components and ATPase

Yin Zhang, Olivier Marcillat, Cecilia R Giulivi, Lars Ernster, Kelvin J A Davies

Research output: Contribution to journalArticle

590 Citations (Scopus)

Abstract

Bovine heart submitochondrial particles (SMP) were exposed to continuous fluxes of hydroxyl radical (OH) alone, superoxide anion radical (O- 2) alone, or mixtures of OH and O- 2, by γ radiolysis in the presence of 100% N2O (OH exposure), 100% O2 + formate (O- 2 exposure), or 100% O2 alone (OH + O- 2 exposure). Hydrogen peroxide effects were studied by addition of pure H2O2. NADH dehydrogenase, NADH oxidase, succinate dehydrogenase, succinate oxidase, and ATPase activities (Vmax) were rapidly inactivated by OH (10% inactivation at 15-40 nmol of OH/mg of SMP protein, 50-90% inactivation at 600 nmol of OH/mg of SMP protein) and by OH + O- 2 (10% inactivation at 20-80 nmol of OH + O- 2/mg of SMP protein, 45-75% inactivation at 600 nmol of OH + O- 2/mg of SMP protein). Importantly, O- 2 was a highly efficient inactivator of NADH dehydrogenase, NADH oxidase, and ATPase (10% inactivation at 20-50 nmol of O- 2/mg of SMP protein, 40% inactivation at 600 nmol of O- 2/mg of SMP protein), a mildly efficient inactivator of succinate dehydrogenase (10% inactivation at 150 nmol of O- 2/mg of SMP protein, 30% inactivation at 600 nmol of O- 2/mg of SMP protein), and a poor inactivator of succinate oxidase (<10% inactivation at 600 nmol of O- 2/mg of SMP protein). H2O2 partially inactivated NADH dehydrogenase, NADH oxidase, and cytochrome oxidase, but even 10% loss of these activities required at least 500-600 nmol of H2O2/mg of SMP protein. Cytochrome oxidase activity (oxygen consumption supported by ascorbate + N,N,N′,N′-tetramethyl-p-phenylenediamine) was remarkably resistant to oxidative inactivation, with less than 20% loss of activity evident even at OH, O- 2, OH + O- 2, or H2O2 concentrations of 600 nmol/mg of SMP protein. Cytochrome c oxidase activity, however (oxidation of, added, ferrocytochrome c), exhibited more than a 40% inactivation at 600 nmol of OH/mg of SMP protein. The OH-dependent inactivations reported above were largely inhibitable by the OH scavenger mannitol. In contrast, the O- 2-dependent inactivations were inhibited by active superoxide dismutase, but not by denatured superoxide dismutase or catalase. Membrane lipid peroxidation was evident with OH exposure but could be prevented by various lipid-soluble antioxidants which did not protect enzymatic activities at all. We conclude that both OH and O- 2 are effective inactivators of specific proteins in the respiratory chain, but that the patterns of inactivation are quite distinct for the two active oxygen species: in contrast, the respiratory chain appears to be far more resistant to H2O2. Generalized lipid peroxidation, although a simple and widely used indicator of oxidative stress, appears to be unrelated to electron transport chain inactivation. The pattern of results presented here may prove useful in probing molecular mechanisms of oxidative damage to mitochondria, and in the development of effective antioxidant strategies.

Original languageEnglish (US)
Pages (from-to)16330-16336
Number of pages7
JournalJournal of Biological Chemistry
Volume265
Issue number27
StatePublished - Sep 25 1990
Externally publishedYes

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Submitochondrial Particles
Electron Transport
Adenosine Triphosphatases
Proteins
NADH Dehydrogenase
Electron Transport Complex IV
formic acid
Succinate Dehydrogenase
Superoxides
Lipid Peroxidation
Superoxide Dismutase
Antioxidants
Lipids
Radiolysis
Mitochondria
Oxidative stress
Mannitol
Membrane Lipids
Cytochromes c
Oxygen Consumption

ASJC Scopus subject areas

  • Biochemistry

Cite this

Zhang, Y., Marcillat, O., Giulivi, C. R., Ernster, L., & Davies, K. J. A. (1990). The oxidative inactivation of mitochondrial electron transport chain components and ATPase. Journal of Biological Chemistry, 265(27), 16330-16336.

The oxidative inactivation of mitochondrial electron transport chain components and ATPase. / Zhang, Yin; Marcillat, Olivier; Giulivi, Cecilia R; Ernster, Lars; Davies, Kelvin J A.

In: Journal of Biological Chemistry, Vol. 265, No. 27, 25.09.1990, p. 16330-16336.

Research output: Contribution to journalArticle

Zhang, Y, Marcillat, O, Giulivi, CR, Ernster, L & Davies, KJA 1990, 'The oxidative inactivation of mitochondrial electron transport chain components and ATPase', Journal of Biological Chemistry, vol. 265, no. 27, pp. 16330-16336.
Zhang, Yin ; Marcillat, Olivier ; Giulivi, Cecilia R ; Ernster, Lars ; Davies, Kelvin J A. / The oxidative inactivation of mitochondrial electron transport chain components and ATPase. In: Journal of Biological Chemistry. 1990 ; Vol. 265, No. 27. pp. 16330-16336.
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abstract = "Bovine heart submitochondrial particles (SMP) were exposed to continuous fluxes of hydroxyl radical (•OH) alone, superoxide anion radical (O- 2) alone, or mixtures of •OH and O- 2, by γ radiolysis in the presence of 100{\%} N2O (•OH exposure), 100{\%} O2 + formate (O- 2 exposure), or 100{\%} O2 alone (•OH + O- 2 exposure). Hydrogen peroxide effects were studied by addition of pure H2O2. NADH dehydrogenase, NADH oxidase, succinate dehydrogenase, succinate oxidase, and ATPase activities (Vmax) were rapidly inactivated by •OH (10{\%} inactivation at 15-40 nmol of •OH/mg of SMP protein, 50-90{\%} inactivation at 600 nmol of •OH/mg of SMP protein) and by •OH + O- 2 (10{\%} inactivation at 20-80 nmol of •OH + O- 2/mg of SMP protein, 45-75{\%} inactivation at 600 nmol of •OH + O- 2/mg of SMP protein). Importantly, O- 2 was a highly efficient inactivator of NADH dehydrogenase, NADH oxidase, and ATPase (10{\%} inactivation at 20-50 nmol of O- 2/mg of SMP protein, 40{\%} inactivation at 600 nmol of O- 2/mg of SMP protein), a mildly efficient inactivator of succinate dehydrogenase (10{\%} inactivation at 150 nmol of O- 2/mg of SMP protein, 30{\%} inactivation at 600 nmol of O- 2/mg of SMP protein), and a poor inactivator of succinate oxidase (<10{\%} inactivation at 600 nmol of O- 2/mg of SMP protein). H2O2 partially inactivated NADH dehydrogenase, NADH oxidase, and cytochrome oxidase, but even 10{\%} loss of these activities required at least 500-600 nmol of H2O2/mg of SMP protein. Cytochrome oxidase activity (oxygen consumption supported by ascorbate + N,N,N′,N′-tetramethyl-p-phenylenediamine) was remarkably resistant to oxidative inactivation, with less than 20{\%} loss of activity evident even at •OH, O- 2, •OH + O- 2, or H2O2 concentrations of 600 nmol/mg of SMP protein. Cytochrome c oxidase activity, however (oxidation of, added, ferrocytochrome c), exhibited more than a 40{\%} inactivation at 600 nmol of •OH/mg of SMP protein. The •OH-dependent inactivations reported above were largely inhibitable by the •OH scavenger mannitol. In contrast, the O- 2-dependent inactivations were inhibited by active superoxide dismutase, but not by denatured superoxide dismutase or catalase. Membrane lipid peroxidation was evident with •OH exposure but could be prevented by various lipid-soluble antioxidants which did not protect enzymatic activities at all. We conclude that both •OH and O- 2 are effective inactivators of specific proteins in the respiratory chain, but that the patterns of inactivation are quite distinct for the two active oxygen species: in contrast, the respiratory chain appears to be far more resistant to H2O2. Generalized lipid peroxidation, although a simple and widely used indicator of oxidative stress, appears to be unrelated to electron transport chain inactivation. The pattern of results presented here may prove useful in probing molecular mechanisms of oxidative damage to mitochondria, and in the development of effective antioxidant strategies.",
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TY - JOUR

T1 - The oxidative inactivation of mitochondrial electron transport chain components and ATPase

AU - Zhang, Yin

AU - Marcillat, Olivier

AU - Giulivi, Cecilia R

AU - Ernster, Lars

AU - Davies, Kelvin J A

PY - 1990/9/25

Y1 - 1990/9/25

N2 - Bovine heart submitochondrial particles (SMP) were exposed to continuous fluxes of hydroxyl radical (•OH) alone, superoxide anion radical (O- 2) alone, or mixtures of •OH and O- 2, by γ radiolysis in the presence of 100% N2O (•OH exposure), 100% O2 + formate (O- 2 exposure), or 100% O2 alone (•OH + O- 2 exposure). Hydrogen peroxide effects were studied by addition of pure H2O2. NADH dehydrogenase, NADH oxidase, succinate dehydrogenase, succinate oxidase, and ATPase activities (Vmax) were rapidly inactivated by •OH (10% inactivation at 15-40 nmol of •OH/mg of SMP protein, 50-90% inactivation at 600 nmol of •OH/mg of SMP protein) and by •OH + O- 2 (10% inactivation at 20-80 nmol of •OH + O- 2/mg of SMP protein, 45-75% inactivation at 600 nmol of •OH + O- 2/mg of SMP protein). Importantly, O- 2 was a highly efficient inactivator of NADH dehydrogenase, NADH oxidase, and ATPase (10% inactivation at 20-50 nmol of O- 2/mg of SMP protein, 40% inactivation at 600 nmol of O- 2/mg of SMP protein), a mildly efficient inactivator of succinate dehydrogenase (10% inactivation at 150 nmol of O- 2/mg of SMP protein, 30% inactivation at 600 nmol of O- 2/mg of SMP protein), and a poor inactivator of succinate oxidase (<10% inactivation at 600 nmol of O- 2/mg of SMP protein). H2O2 partially inactivated NADH dehydrogenase, NADH oxidase, and cytochrome oxidase, but even 10% loss of these activities required at least 500-600 nmol of H2O2/mg of SMP protein. Cytochrome oxidase activity (oxygen consumption supported by ascorbate + N,N,N′,N′-tetramethyl-p-phenylenediamine) was remarkably resistant to oxidative inactivation, with less than 20% loss of activity evident even at •OH, O- 2, •OH + O- 2, or H2O2 concentrations of 600 nmol/mg of SMP protein. Cytochrome c oxidase activity, however (oxidation of, added, ferrocytochrome c), exhibited more than a 40% inactivation at 600 nmol of •OH/mg of SMP protein. The •OH-dependent inactivations reported above were largely inhibitable by the •OH scavenger mannitol. In contrast, the O- 2-dependent inactivations were inhibited by active superoxide dismutase, but not by denatured superoxide dismutase or catalase. Membrane lipid peroxidation was evident with •OH exposure but could be prevented by various lipid-soluble antioxidants which did not protect enzymatic activities at all. We conclude that both •OH and O- 2 are effective inactivators of specific proteins in the respiratory chain, but that the patterns of inactivation are quite distinct for the two active oxygen species: in contrast, the respiratory chain appears to be far more resistant to H2O2. Generalized lipid peroxidation, although a simple and widely used indicator of oxidative stress, appears to be unrelated to electron transport chain inactivation. The pattern of results presented here may prove useful in probing molecular mechanisms of oxidative damage to mitochondria, and in the development of effective antioxidant strategies.

AB - Bovine heart submitochondrial particles (SMP) were exposed to continuous fluxes of hydroxyl radical (•OH) alone, superoxide anion radical (O- 2) alone, or mixtures of •OH and O- 2, by γ radiolysis in the presence of 100% N2O (•OH exposure), 100% O2 + formate (O- 2 exposure), or 100% O2 alone (•OH + O- 2 exposure). Hydrogen peroxide effects were studied by addition of pure H2O2. NADH dehydrogenase, NADH oxidase, succinate dehydrogenase, succinate oxidase, and ATPase activities (Vmax) were rapidly inactivated by •OH (10% inactivation at 15-40 nmol of •OH/mg of SMP protein, 50-90% inactivation at 600 nmol of •OH/mg of SMP protein) and by •OH + O- 2 (10% inactivation at 20-80 nmol of •OH + O- 2/mg of SMP protein, 45-75% inactivation at 600 nmol of •OH + O- 2/mg of SMP protein). Importantly, O- 2 was a highly efficient inactivator of NADH dehydrogenase, NADH oxidase, and ATPase (10% inactivation at 20-50 nmol of O- 2/mg of SMP protein, 40% inactivation at 600 nmol of O- 2/mg of SMP protein), a mildly efficient inactivator of succinate dehydrogenase (10% inactivation at 150 nmol of O- 2/mg of SMP protein, 30% inactivation at 600 nmol of O- 2/mg of SMP protein), and a poor inactivator of succinate oxidase (<10% inactivation at 600 nmol of O- 2/mg of SMP protein). H2O2 partially inactivated NADH dehydrogenase, NADH oxidase, and cytochrome oxidase, but even 10% loss of these activities required at least 500-600 nmol of H2O2/mg of SMP protein. Cytochrome oxidase activity (oxygen consumption supported by ascorbate + N,N,N′,N′-tetramethyl-p-phenylenediamine) was remarkably resistant to oxidative inactivation, with less than 20% loss of activity evident even at •OH, O- 2, •OH + O- 2, or H2O2 concentrations of 600 nmol/mg of SMP protein. Cytochrome c oxidase activity, however (oxidation of, added, ferrocytochrome c), exhibited more than a 40% inactivation at 600 nmol of •OH/mg of SMP protein. The •OH-dependent inactivations reported above were largely inhibitable by the •OH scavenger mannitol. In contrast, the O- 2-dependent inactivations were inhibited by active superoxide dismutase, but not by denatured superoxide dismutase or catalase. Membrane lipid peroxidation was evident with •OH exposure but could be prevented by various lipid-soluble antioxidants which did not protect enzymatic activities at all. We conclude that both •OH and O- 2 are effective inactivators of specific proteins in the respiratory chain, but that the patterns of inactivation are quite distinct for the two active oxygen species: in contrast, the respiratory chain appears to be far more resistant to H2O2. Generalized lipid peroxidation, although a simple and widely used indicator of oxidative stress, appears to be unrelated to electron transport chain inactivation. The pattern of results presented here may prove useful in probing molecular mechanisms of oxidative damage to mitochondria, and in the development of effective antioxidant strategies.

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