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Cited by 231Redox biology and oxidative stressSulfur Compounds in BiologyAntioxidant Activity and Oxidative Stress

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Cysteine residues exposed on protein surfaces are the dominant intramitochondrial thiol and may protect against oxidative damage

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Requejo, Raquel, Hurd, Thomas R., Costa, Nikola J., Murphy, Michael P.·2010·The FEBS Journal
DOI 10.1111/j.1742-4658.2010.07576.xPMC PMC2847196
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Abstract

Cysteine plays a number of important roles in protecting the cell from oxidative damage through its thiol functional group. These defensive functions are generally considered to be carried out by the low molecular weight thiol glutathione and by cysteine residues in the active sites of proteins such as thioredoxin and peroxiredoxin. In addition, there are thiols exposed on protein surfaces that are not directly involved with protein function, although they can interact with the intracellular environment. In the present study, in subcellular fractions prepared from rat liver or heart, we show that the quantitatively dominant free thiols are those of cysteine residues exposed on protein surfaces and not those carried by glutathione. Within the mitochondrial matrix, the concentration of exposed protein thiols is 60–90 m m, which is approximately 26‐fold higher than the glutathione concentration in that compartment. This suggests that exposed protein thiols are of greater importance than glutathione for nonenzyme catalysed reactions of thiols with reactive oxygen and nitrogen species and with electrophiles within the cell. One such antioxidant role for exposed protein thiols may be to prevent protein oxidative damage. In the present study, in mitochondrial membranes and in complex I, we show that exposed protein thiols protect against tyrosine nitration and protein dysfunction caused by peroxynitrite. Therefore, exposed protein thiols are the dominant free thiol within the cell and may play a critical role in intracellular antioxidant defences against oxidative damage.

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Fig. 1 Total and exposed protein thiols and GSH in liver and heart tissue homogenates and mitochondria. (A, B) Total and exposed protein thiols in sequential supernatants from 3000 g, 10 000 g and 100 000 g centrifugations, and from a mitochondrial fraction, isolated from liver (A) and heart (B) tissue homogenates. **P < 0.01 for comparison of total and exposed thiols by Student’s t-test. (C, D) Total GSH equivalents, GSH and 2× GSSG, in sequential supernatants from 3000 g, 10 000 g and 100 000 g centrifugations, and from a mitochondrial fraction, isolated from liver (C) and heart (D) tissue homogenates. The percentages above the data bars indicate the total GSH content of the fraction as a percentage of its exposed protein thiol content. (E, F) Total and exposed thiols in membrane and matrix fractions from liver (E) or heart (F) mitochondria. Mitochondria (5 mg·mL−1 protein) were suspended in KCl buffer, pelleted by centrifugation and separated into membrane and matrix fractions and then exposed and total protein thiols were measured. (G) Exposed mitochondrial protein thiols ± the thiol alkylating agent AMS. Mitochondria (5 mg·mL−1 protein) were incubated in KCl buffer ± AMS (100 μm) for 10 min at 30 °C. Samples were then centrifuged and exposed protein thiols were measured. (H) GSH content of rat liver and heart mitochondria. Mitochondria (5 mg·mL−1 protein) were incubated in KCl buffer for 10 min at 30 °C and the GSH and GSSG contents measured. All data are the mean ± SD of three independent experiments.
Fig. 1 Total and exposed protein thiols and GSH in liver and heart tissue homogenates and mitochondria. (A, B) Total and exposed protein thiols in sequential supernatants from 3000 g, 10 000 g and 100 000 g centrifugations, and from a mitochondrial fraction, isolated from liver (A) and heart (B) tissue homogenates. **P < 0.01 for comparison of total and exposed thiols by Student’s t-test. (C, D) Total GSH equivalents, GSH and 2× GSSG, in sequential supernatants from 3000 g, 10 000 g and 100 000 g centrifugations, and from a mitochondrial fraction, isolated from liver (C) and heart (D) tissue homogenates. The percentages above the data bars indicate the total GSH content of the fraction as a percentage of its exposed protein thiol content. (E, F) Total and exposed thiols in membrane and matrix fractions from liver (E) or heart (F) mitochondria. Mitochondria (5 mg·mL−1 protein) were suspended in KCl buffer, pelleted by centrifugation and separated into membrane and matrix fractions and then exposed and total protein thiols were measured. (G) Exposed mitochondrial protein thiols ± the thiol alkylating agent AMS. Mitochondria (5 mg·mL−1 protein) were incubated in KCl buffer ± AMS (100 μm) for 10 min at 30 °C. Samples were then centrifuged and exposed protein thiols were measured. (H) GSH content of rat liver and heart mitochondria. Mitochondria (5 mg·mL−1 protein) were incubated in KCl buffer for 10 min at 30 °C and the GSH and GSSG contents measured. All data are the mean ± SD of three independent experiments.CC BY · PubMed Central
Fig. 2 Exposed protein thiols and GSH in oxidatively stressed mitochondria. (A–D) Effect of diamide on exposed protein thiols, protein-GSH mixed disulfides and GSH. Mitochondria (5 mg·mL−1 protein) from the liver (A, C) or heart (B, D) were incubated with diamide for 5 min at 37 °C. The values after the Δ in (C) and (D) are the actual and the percentage changes in protein thiols relative to controls. (E) Effects of oxidants and dithiothreitol on exposed mitochondrial protein thiols. Liver mitochondria (5 mg·mL−1 protein) were incubated for 5 min with 0.5 mm ONOO−, tBHP or diamide and exposed protein thiols measured. For some incubations, the mitochondria were incubated with 1 mm dithiothreitol before measurement of protein thiols. The values after the Δ in (C) and (D) are the actual and the percentage changes in protein thiols relative to controls. (F) Reduction of mitochondrial thiols after oxidative stress. Liver mitochondria (5 mg·mL−1 protein) were incubated with either carrier, 0.5 mm tBHP, ONOO− or diamide for 10 min. Next, mitochondria were pelleted by centrifugation and resuspended in medium without oxidant. The exposed protein thiols were measured as a percentage of parallel control incubations that had undergone the same isolation and resuspension procedures but without exposure to oxidant. All data are the mean ± SD of three experiments: *P < 0.05, **P < 0.01 relative to controls by Student’s t-test. DTT, dithiothreitol.
Fig. 2 Exposed protein thiols and GSH in oxidatively stressed mitochondria. (A–D) Effect of diamide on exposed protein thiols, protein-GSH mixed disulfides and GSH. Mitochondria (5 mg·mL−1 protein) from the liver (A, C) or heart (B, D) were incubated with diamide for 5 min at 37 °C. The values after the Δ in (C) and (D) are the actual and the percentage changes in protein thiols relative to controls. (E) Effects of oxidants and dithiothreitol on exposed mitochondrial protein thiols. Liver mitochondria (5 mg·mL−1 protein) were incubated for 5 min with 0.5 mm ONOO−, tBHP or diamide and exposed protein thiols measured. For some incubations, the mitochondria were incubated with 1 mm dithiothreitol before measurement of protein thiols. The values after the Δ in (C) and (D) are the actual and the percentage changes in protein thiols relative to controls. (F) Reduction of mitochondrial thiols after oxidative stress. Liver mitochondria (5 mg·mL−1 protein) were incubated with either carrier, 0.5 mm tBHP, ONOO− or diamide for 10 min. Next, mitochondria were pelleted by centrifugation and resuspended in medium without oxidant. The exposed protein thiols were measured as a percentage of parallel control incubations that had undergone the same isolation and resuspension procedures but without exposure to oxidant. All data are the mean ± SD of three experiments: *P < 0.05, **P < 0.01 relative to controls by Student’s t-test. DTT, dithiothreitol.CC BY · PubMed Central
Fig. 3 Effect of blocking exposed protein thiols with N-ethylmaleimide on nitration by ONOO− of mitochondrial membrane proteins. (A–D) Mitochondrial membranes were incubated (20 mg·mL−1 protein) at 37 °C in membrane buffer with either no additions or after pre-treatment with 1 mmN-ethylmaleimide for 10 min. Next, the membranes were exposed to different doses of ONOO−, or decomposed ONOO− for the control incubations, for 5 min. (A) After incubation exposed protein thiols were measured. Data are the mean ± SD of three experiments. ***P < 0.001 by Student’s t-test. (B) After incubation mitochondrial membranes (75 μg of protein) was separated by SDS-PAGE and immunblotted to detect 3-nitrotyrosine residues. (C) After incubation, mitochondrial membranes (50 μg of protein) was separated by SDS-PAGE and immunoblotted to detect 3-nitrotyrosine residues. (D) Membranes were incubated as above but in the presence of DMPO (100 mm). After incubation, mitochondrial membranes (75 μg of protein) was separated by SDS-PAGE and immunoblotted to detect DMPO protein adducts. (E, F) Rate of decay of ONOO−. (E) The decomposition of ONOO− (1 mm) was monitored by measuring A302 after its addition to a rapidly stirred suspension of membranes (1 mg·mL−1 protein) incubated as described above in presence or absence of 1 mmN-ethylmaleimide. (F) Rate constants for decomposition of ONOO− in controls or samples containing mitochondrial membranes, with or without N-ethylmaleimide. Data are the mean ± SD of three experiments. DTT, dithiothreitol; NEM, N-ethylmaleimide.
Fig. 3 Effect of blocking exposed protein thiols with N-ethylmaleimide on nitration by ONOO− of mitochondrial membrane proteins. (A–D) Mitochondrial membranes were incubated (20 mg·mL−1 protein) at 37 °C in membrane buffer with either no additions or after pre-treatment with 1 mmN-ethylmaleimide for 10 min. Next, the membranes were exposed to different doses of ONOO−, or decomposed ONOO− for the control incubations, for 5 min. (A) After incubation exposed protein thiols were measured. Data are the mean ± SD of three experiments. ***P < 0.001 by Student’s t-test. (B) After incubation mitochondrial membranes (75 μg of protein) was separated by SDS-PAGE and immunblotted to detect 3-nitrotyrosine residues. (C) After incubation, mitochondrial membranes (50 μg of protein) was separated by SDS-PAGE and immunoblotted to detect 3-nitrotyrosine residues. (D) Membranes were incubated as above but in the presence of DMPO (100 mm). After incubation, mitochondrial membranes (75 μg of protein) was separated by SDS-PAGE and immunoblotted to detect DMPO protein adducts. (E, F) Rate of decay of ONOO−. (E) The decomposition of ONOO− (1 mm) was monitored by measuring A302 after its addition to a rapidly stirred suspension of membranes (1 mg·mL−1 protein) incubated as described above in presence or absence of 1 mmN-ethylmaleimide. (F) Rate constants for decomposition of ONOO− in controls or samples containing mitochondrial membranes, with or without N-ethylmaleimide. Data are the mean ± SD of three experiments. DTT, dithiothreitol; NEM, N-ethylmaleimide.CC BY · PubMed Central
Fig. 4 Nitration and inhibition of complex I by ONOO−. (A) Mitochondrial membranes were incubated as described in Fig. 3A–D with the indicated concentrations of ONOO− or of decomposed ONOO− for controls. Next, membrane samples (∼ 150 μg of protein per lane) were separated by BN-PAGE, the complex I band excised and further separated by SDS-PAGE and then immunoblotted using an antibody against 3-nitrotyrosine. The blot was reprobed using antisera against the 75 kDa, 51 kDa or 23 kDa complex I subunits and one lane of this is shown. (B) Isolated complex I (25 μg) was incubated in 50 μL of KCl buffer at 37 °C for 10 min ± N-ethylmaleimide, then the indicated concentrations of ONOO−, or decomposed ONOO− for controls, were added and the samples were processed 5 min later. For this, 300 μL of ethanol was added and, after overnight incubation at −20 °C, protein was pelleted, suspended in loading buffer, and 10 μg of protein was separated by SDS-PAGE and immunblotted using an antibody against 3-nitrotyrosine. The blot was reprobed using antisera against the 75 kDa and 51 kDa complex I subunits and one lane of this is shown. (C) The activity of complex I measured as NADH:HAR oxidoreductase activity in membranes and isolated complex I. Data are the mean ± SD of three independent measurements and are the percentage of parallel control measurements. Data are the mean ± SD of three measurements (*P < 0.05, **P < 0.01, ***P < 0.001). NEM, N-ethylmaleimide.
Fig. 4 Nitration and inhibition of complex I by ONOO−. (A) Mitochondrial membranes were incubated as described in Fig. 3A–D with the indicated concentrations of ONOO− or of decomposed ONOO− for controls. Next, membrane samples (∼ 150 μg of protein per lane) were separated by BN-PAGE, the complex I band excised and further separated by SDS-PAGE and then immunoblotted using an antibody against 3-nitrotyrosine. The blot was reprobed using antisera against the 75 kDa, 51 kDa or 23 kDa complex I subunits and one lane of this is shown. (B) Isolated complex I (25 μg) was incubated in 50 μL of KCl buffer at 37 °C for 10 min ± N-ethylmaleimide, then the indicated concentrations of ONOO−, or decomposed ONOO− for controls, were added and the samples were processed 5 min later. For this, 300 μL of ethanol was added and, after overnight incubation at −20 °C, protein was pelleted, suspended in loading buffer, and 10 μg of protein was separated by SDS-PAGE and immunblotted using an antibody against 3-nitrotyrosine. The blot was reprobed using antisera against the 75 kDa and 51 kDa complex I subunits and one lane of this is shown. (C) The activity of complex I measured as NADH:HAR oxidoreductase activity in membranes and isolated complex I. Data are the mean ± SD of three independent measurements and are the percentage of parallel control measurements. Data are the mean ± SD of three measurements (*P < 0.05, **P < 0.01, ***P < 0.001). NEM, N-ethylmaleimide.CC BY · PubMed Central
Fig. 5 Glutathionylation of exposed protein thiyl radicals. Mitochondrial membranes (1 mg·mL−1 protein) were incubated in 1 mL of membrane buffer containing 2 mm succinate and 4 μg of rotenone within a rapidly stirred O2 electrode chamber. (A) Anaerobic incubation of mitochondrial membranes. An O2 electrode trace of a typical mitochondrial membrane experiment is shown. Respiration by the membranes consumes all of the O2, causing the incubation to become anaerobic. The points corresponding to those at which ONOO− and [3H]GSH were added and where the samples were taken for analysis for the experiment in (D) are indicated on the trace. (B) Time course of ONOO− decay. Mitochondrial membranes (1 mg·mL−1 protein) were stirred rapidly in a 3 mL sealed, anaerobic cuvette and, where indicated, 1 mm ONOO− was added and its decay followed at 302 nm. (C) Stability of protein radicals produced by the treatment with ONOO−. Mitochondrial membranes were incubated at room temperature ± N-ethylmaleimide (NEM) (1 mm) for 10 min under anaerobic conditions. Next, ONOO− (0.5 mm) was added at various times, and then DMPO (100 mm) was added. The membrane proteins (150 μg of protein) were separated by SDS-PAGE and immunoblotted to detect DMPO-protein adducts. (D) Mitochondrial membranes were incubated anaerobically as above. Next, the indicated concentrations of ONOO− were added, 30 s later 100 μm [3H]GSH was added and, 2 min later, membranes were isolated, treated ± dithithreitol and the content of [3H]GSH determined by scintillation counting. Data are the mean ± SD of three experiments. *P < 0.05 by Student’s t-test. DTT, dithiothreitol.
Fig. 5 Glutathionylation of exposed protein thiyl radicals. Mitochondrial membranes (1 mg·mL−1 protein) were incubated in 1 mL of membrane buffer containing 2 mm succinate and 4 μg of rotenone within a rapidly stirred O2 electrode chamber. (A) Anaerobic incubation of mitochondrial membranes. An O2 electrode trace of a typical mitochondrial membrane experiment is shown. Respiration by the membranes consumes all of the O2, causing the incubation to become anaerobic. The points corresponding to those at which ONOO− and [3H]GSH were added and where the samples were taken for analysis for the experiment in (D) are indicated on the trace. (B) Time course of ONOO− decay. Mitochondrial membranes (1 mg·mL−1 protein) were stirred rapidly in a 3 mL sealed, anaerobic cuvette and, where indicated, 1 mm ONOO− was added and its decay followed at 302 nm. (C) Stability of protein radicals produced by the treatment with ONOO−. Mitochondrial membranes were incubated at room temperature ± N-ethylmaleimide (NEM) (1 mm) for 10 min under anaerobic conditions. Next, ONOO− (0.5 mm) was added at various times, and then DMPO (100 mm) was added. The membrane proteins (150 μg of protein) were separated by SDS-PAGE and immunoblotted to detect DMPO-protein adducts. (D) Mitochondrial membranes were incubated anaerobically as above. Next, the indicated concentrations of ONOO− were added, 30 s later 100 μm [3H]GSH was added and, 2 min later, membranes were isolated, treated ± dithithreitol and the content of [3H]GSH determined by scintillation counting. Data are the mean ± SD of three experiments. *P < 0.05 by Student’s t-test. DTT, dithiothreitol.CC BY · PubMed Central
Fig. 6 Modes of protection against oxidative damage by exposed protein thiols. The three panels show the various ways in which exposed protein thiols can protect against oxidative damage. (A) Modes of recycling of exposed protein thiols after oxidation. A schematic protein (shaded) is shown with two exposed thiols. Oxidation by ROS can generate a sulfenic acid (RSOH) or a thiyl radical. These can be irreversibly oxidized to higher thiol oxidation states (RSOnH). The sulfenic acid can be converted to an intramolecular disulfide, or form a mixed disulfide with GSH. The thiyl radical can form a radical anion intramolecular disulfide, or a mixed disulfide with GSH. These can lose an electron to O2 to form superoxide. The mixed disulfide thus formed can be recycled to a thiol by the action of GSH and Grx, whereas an intramolecular disulfide can be recycled by Trx. (B) Intramolecular electron transfer from a thiol to a tyrosyl radical. ROS generates a tyrosyl radical on a tyrosine residue, which is then is reduced by an adjacent thiol to generate a thiyl radical. The thiyl radical can be recycled back to a thiol by the mechanisms outlined in (A). (C) Role of NO in preventing protein oxidative damage. The thiyl radical generated by ROS can react rapidly with NO to generate a S-nitrosothiol. This will decrease the extent of irreversible oxidation of the thiol. The S-nitrosothiol can then be recycled back to a disulfide as shown.
Fig. 6 Modes of protection against oxidative damage by exposed protein thiols. The three panels show the various ways in which exposed protein thiols can protect against oxidative damage. (A) Modes of recycling of exposed protein thiols after oxidation. A schematic protein (shaded) is shown with two exposed thiols. Oxidation by ROS can generate a sulfenic acid (RSOH) or a thiyl radical. These can be irreversibly oxidized to higher thiol oxidation states (RSOnH). The sulfenic acid can be converted to an intramolecular disulfide, or form a mixed disulfide with GSH. The thiyl radical can form a radical anion intramolecular disulfide, or a mixed disulfide with GSH. These can lose an electron to O2 to form superoxide. The mixed disulfide thus formed can be recycled to a thiol by the action of GSH and Grx, whereas an intramolecular disulfide can be recycled by Trx. (B) Intramolecular electron transfer from a thiol to a tyrosyl radical. ROS generates a tyrosyl radical on a tyrosine residue, which is then is reduced by an adjacent thiol to generate a thiyl radical. The thiyl radical can be recycled back to a thiol by the mechanisms outlined in (A). (C) Role of NO in preventing protein oxidative damage. The thiyl radical generated by ROS can react rapidly with NO to generate a S-nitrosothiol. This will decrease the extent of irreversible oxidation of the thiol. The S-nitrosothiol can then be recycled back to a disulfide as shown.CC BY · PubMed Central

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Metadata

Authors
Requejo, R., Hurd, T. R., Costa, N. J., & Murphy, M. P.
Affiliations
MRC Mitochondrial Biology Unit (GB)
ORCID
Venue
The FEBS Journal
Publisher
Wiley
Published
2010-02-24
Volume
277
Issue
6
Pages
1465-1480
ISSN
  • 1742-464X (ISSN-L)
  • 1742-4658
DOI
10.1111/j.1742-4658.2010.07576.x
PMC
PMC2847196
OpenAlex
W1524076404
Cited by
231
References
52
Citations by year
2026: 7 · 2025: 7 · 2024: 14 · 2023: 12 · 2022: 15 · 2021: 13 · 2020: 11 · 2019: 13 · 2018: 20 · 2017: 11 · 2016: 14 · 2015: 18 · 2014: 24 · 2013: 16 · 2012: 14
FWCI
6.77
Type
journal article
Language
en
Open access
Licence
Locations
  • FEBS Journal (publishedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • Ministerio de Ciencia y Tecnología
  • Medical Research Council
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Sulfur Compounds in Biology
  • Health Sciences › Medicine › Biochemistry (medical) › Antioxidant Activity and Oxidative Stress
Keywords
cysteine residues, mitochondrial matrix, glutathione, oxidative damage, antioxidant defense, peroxynitrite, tyrosine nitration, protein oxidation, mitochondrial complex I, reactive oxygen species, reactive nitrogen species, electrophiles, rat liver, rat heart
MeSH terms
Animals, Cells, Cultured, Cysteine, Glutathione, Membrane Proteins, Mitochondria, Heart, Mitochondria, Liver, Sulfhydryl Compounds, Oxidative Stress, Rats

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