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Malfunctioning of the iron-sulfur cluster assembly machinery in Saccharomyces cerevisiae produces oxidative stress via an iron-dependent mechanism, causing dysfunction in respiratory complexes.

Gomez M, Pérez-Gallardo RV, Sánchez LA, Díaz-Pérez AL, Cortés-Rojo C, Meza Carmen V, Saavedra-Molina A, Lara-Romero J, Jiménez-Sandoval S, Rodríguez F, Rodríguez-Zavala JS, Campos-García J - PLoS ONE (2014)

Bottom Line: Our study suggests that the increment in free Fe2+ associated with ROS generation may have originated from mitochondria, probably Fe-S cluster proteins, under both normal and oxidative stress conditions, suggesting that Fe-S cluster anabolism is affected.Raman spectroscopy analysis and immunoblotting indicated that in mitochondria from SSQ1 and ISA1 mutants, the content of [Fe-S] centers was decreased, as was formation of Rieske protein-dependent supercomplex III2IV2, but this was not observed in the iron-deficient ATX1 and MRS4 mutants.These results confirm that the ISC system plays important roles in iron homeostasis, ROS stress, and in assembly of supercomplexes III2IV2 and III2IV1, thus affecting the functionality of the respiratory chain.

View Article: PubMed Central - PubMed

Affiliation: Lab. Biotecnología Microbiana, Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacán, México.

ABSTRACT
Biogenesis and recycling of iron-sulfur (Fe-S) clusters play important roles in the iron homeostasis mechanisms involved in mitochondrial function. In Saccharomyces cerevisiae, the Fe-S clusters are assembled into apoproteins by the iron-sulfur cluster machinery (ISC). The aim of the present study was to determine the effects of ISC gene deletion and consequent iron release under oxidative stress conditions on mitochondrial functionality in S. cerevisiae. Reactive oxygen species (ROS) generation, caused by H2O2, menadione, or ethanol, was associated with a loss of iron homeostasis and exacerbated by ISC system dysfunction. ISC mutants showed increased free Fe2+ content, exacerbated by ROS-inducers, causing an increase in ROS, which was decreased by the addition of an iron chelator. Our study suggests that the increment in free Fe2+ associated with ROS generation may have originated from mitochondria, probably Fe-S cluster proteins, under both normal and oxidative stress conditions, suggesting that Fe-S cluster anabolism is affected. Raman spectroscopy analysis and immunoblotting indicated that in mitochondria from SSQ1 and ISA1 mutants, the content of [Fe-S] centers was decreased, as was formation of Rieske protein-dependent supercomplex III2IV2, but this was not observed in the iron-deficient ATX1 and MRS4 mutants. In addition, the activity of complexes II and IV from the electron transport chain (ETC) was impaired or totally abolished in SSQ1 and ISA1 mutants. These results confirm that the ISC system plays important roles in iron homeostasis, ROS stress, and in assembly of supercomplexes III2IV2 and III2IV1, thus affecting the functionality of the respiratory chain.

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Analysis of mitochondrial Fe–S-containing proteins in S. cerevisiae ISC mutants.Cell extracts of yeast cultures grown in YPD to the late exponential growth phase were used to isolate mitochondria, as described in the Materials and Methods section. A) Raman scattering spectra of the mitochondria isolated from S. cerevisiae ISC mutants. Raman spectra were recorded at a laser excitation of 632.8 nm with 30 mW. Each spectrum is the average of scans recorded over 60 sec, using photon counting at 0.5 cm−1 increment spectral resolution. Bands corresponding to the [2Fe–2S] and [4Fe–4S] clusters are indicated with arrows [26]. B) Enzymatic activity of cis-aconitase was determined in mitochondrial suspensions as described in the Materials and Methods. Values are the mean of three independent experiments. SE values are indicated as bars (n = 3), one-way ANOVA with Tukey's post-hoc test was used to compare yeast strains, and significant differences (p<0.05) are indicated with different lowercase letters.
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pone-0111585-g006: Analysis of mitochondrial Fe–S-containing proteins in S. cerevisiae ISC mutants.Cell extracts of yeast cultures grown in YPD to the late exponential growth phase were used to isolate mitochondria, as described in the Materials and Methods section. A) Raman scattering spectra of the mitochondria isolated from S. cerevisiae ISC mutants. Raman spectra were recorded at a laser excitation of 632.8 nm with 30 mW. Each spectrum is the average of scans recorded over 60 sec, using photon counting at 0.5 cm−1 increment spectral resolution. Bands corresponding to the [2Fe–2S] and [4Fe–4S] clusters are indicated with arrows [26]. B) Enzymatic activity of cis-aconitase was determined in mitochondrial suspensions as described in the Materials and Methods. Values are the mean of three independent experiments. SE values are indicated as bars (n = 3), one-way ANOVA with Tukey's post-hoc test was used to compare yeast strains, and significant differences (p<0.05) are indicated with different lowercase letters.

Mentions: The above results led us to hypothesize that the increment in free iron levels induced by oxidative stress and ethanol and exacerbated by ISC system dysfunction arise partially from iron sources such as proteins containing Fe–S centers, including complexes II and III of the ETC. Raman spectroscopy analysis has been used as an analytical tool for determination of Fe–S species and contents; therefore, this technique was utilized to determine the Fe–S content in mitochondria isolated from ISC mutants or the iron-transport defective mutants atx1Δ and mrs4Δ grown in YPD. Signal intensities in the interval 200–700 cm−1 at 632.8 nm in the Raman spectra are in agreement with signals corresponding to photonic emission, characteristics of previously described [2Fe–2S] and [4Fe–4S] centers [26], [33]. In our system, mitochondria from S. cerevisiae clearly showed Raman signals in stretching regions of peaks at 345–365, 390–440, 460–480, 490–500, 510–520, and 640–660 cm−1 (Fig. 6a). The intensity of Raman signals of the Fe–S centers were clearly diminished in mitochondria from ssq1Δ and isa1Δ mutants compared to WT mitochondria, whereas in grx5Δ mutants, the signal peaks showed increased intensities. Interestingly, the iron deficient atx1Δ and mrs4Δ mutants showed peaks intensities higher than those of the WT. These results indicate that the amount of mitochondrial Fe–S center signals were diminished in mitochondria from ssq1Δ and isa1Δ mutants, but were overproduced in grx5Δ, atx1Δ, and mrs4Δ mutants.


Malfunctioning of the iron-sulfur cluster assembly machinery in Saccharomyces cerevisiae produces oxidative stress via an iron-dependent mechanism, causing dysfunction in respiratory complexes.

Gomez M, Pérez-Gallardo RV, Sánchez LA, Díaz-Pérez AL, Cortés-Rojo C, Meza Carmen V, Saavedra-Molina A, Lara-Romero J, Jiménez-Sandoval S, Rodríguez F, Rodríguez-Zavala JS, Campos-García J - PLoS ONE (2014)

Analysis of mitochondrial Fe–S-containing proteins in S. cerevisiae ISC mutants.Cell extracts of yeast cultures grown in YPD to the late exponential growth phase were used to isolate mitochondria, as described in the Materials and Methods section. A) Raman scattering spectra of the mitochondria isolated from S. cerevisiae ISC mutants. Raman spectra were recorded at a laser excitation of 632.8 nm with 30 mW. Each spectrum is the average of scans recorded over 60 sec, using photon counting at 0.5 cm−1 increment spectral resolution. Bands corresponding to the [2Fe–2S] and [4Fe–4S] clusters are indicated with arrows [26]. B) Enzymatic activity of cis-aconitase was determined in mitochondrial suspensions as described in the Materials and Methods. Values are the mean of three independent experiments. SE values are indicated as bars (n = 3), one-way ANOVA with Tukey's post-hoc test was used to compare yeast strains, and significant differences (p<0.05) are indicated with different lowercase letters.
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pone-0111585-g006: Analysis of mitochondrial Fe–S-containing proteins in S. cerevisiae ISC mutants.Cell extracts of yeast cultures grown in YPD to the late exponential growth phase were used to isolate mitochondria, as described in the Materials and Methods section. A) Raman scattering spectra of the mitochondria isolated from S. cerevisiae ISC mutants. Raman spectra were recorded at a laser excitation of 632.8 nm with 30 mW. Each spectrum is the average of scans recorded over 60 sec, using photon counting at 0.5 cm−1 increment spectral resolution. Bands corresponding to the [2Fe–2S] and [4Fe–4S] clusters are indicated with arrows [26]. B) Enzymatic activity of cis-aconitase was determined in mitochondrial suspensions as described in the Materials and Methods. Values are the mean of three independent experiments. SE values are indicated as bars (n = 3), one-way ANOVA with Tukey's post-hoc test was used to compare yeast strains, and significant differences (p<0.05) are indicated with different lowercase letters.
Mentions: The above results led us to hypothesize that the increment in free iron levels induced by oxidative stress and ethanol and exacerbated by ISC system dysfunction arise partially from iron sources such as proteins containing Fe–S centers, including complexes II and III of the ETC. Raman spectroscopy analysis has been used as an analytical tool for determination of Fe–S species and contents; therefore, this technique was utilized to determine the Fe–S content in mitochondria isolated from ISC mutants or the iron-transport defective mutants atx1Δ and mrs4Δ grown in YPD. Signal intensities in the interval 200–700 cm−1 at 632.8 nm in the Raman spectra are in agreement with signals corresponding to photonic emission, characteristics of previously described [2Fe–2S] and [4Fe–4S] centers [26], [33]. In our system, mitochondria from S. cerevisiae clearly showed Raman signals in stretching regions of peaks at 345–365, 390–440, 460–480, 490–500, 510–520, and 640–660 cm−1 (Fig. 6a). The intensity of Raman signals of the Fe–S centers were clearly diminished in mitochondria from ssq1Δ and isa1Δ mutants compared to WT mitochondria, whereas in grx5Δ mutants, the signal peaks showed increased intensities. Interestingly, the iron deficient atx1Δ and mrs4Δ mutants showed peaks intensities higher than those of the WT. These results indicate that the amount of mitochondrial Fe–S center signals were diminished in mitochondria from ssq1Δ and isa1Δ mutants, but were overproduced in grx5Δ, atx1Δ, and mrs4Δ mutants.

Bottom Line: Our study suggests that the increment in free Fe2+ associated with ROS generation may have originated from mitochondria, probably Fe-S cluster proteins, under both normal and oxidative stress conditions, suggesting that Fe-S cluster anabolism is affected.Raman spectroscopy analysis and immunoblotting indicated that in mitochondria from SSQ1 and ISA1 mutants, the content of [Fe-S] centers was decreased, as was formation of Rieske protein-dependent supercomplex III2IV2, but this was not observed in the iron-deficient ATX1 and MRS4 mutants.These results confirm that the ISC system plays important roles in iron homeostasis, ROS stress, and in assembly of supercomplexes III2IV2 and III2IV1, thus affecting the functionality of the respiratory chain.

View Article: PubMed Central - PubMed

Affiliation: Lab. Biotecnología Microbiana, Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacán, México.

ABSTRACT
Biogenesis and recycling of iron-sulfur (Fe-S) clusters play important roles in the iron homeostasis mechanisms involved in mitochondrial function. In Saccharomyces cerevisiae, the Fe-S clusters are assembled into apoproteins by the iron-sulfur cluster machinery (ISC). The aim of the present study was to determine the effects of ISC gene deletion and consequent iron release under oxidative stress conditions on mitochondrial functionality in S. cerevisiae. Reactive oxygen species (ROS) generation, caused by H2O2, menadione, or ethanol, was associated with a loss of iron homeostasis and exacerbated by ISC system dysfunction. ISC mutants showed increased free Fe2+ content, exacerbated by ROS-inducers, causing an increase in ROS, which was decreased by the addition of an iron chelator. Our study suggests that the increment in free Fe2+ associated with ROS generation may have originated from mitochondria, probably Fe-S cluster proteins, under both normal and oxidative stress conditions, suggesting that Fe-S cluster anabolism is affected. Raman spectroscopy analysis and immunoblotting indicated that in mitochondria from SSQ1 and ISA1 mutants, the content of [Fe-S] centers was decreased, as was formation of Rieske protein-dependent supercomplex III2IV2, but this was not observed in the iron-deficient ATX1 and MRS4 mutants. In addition, the activity of complexes II and IV from the electron transport chain (ETC) was impaired or totally abolished in SSQ1 and ISA1 mutants. These results confirm that the ISC system plays important roles in iron homeostasis, ROS stress, and in assembly of supercomplexes III2IV2 and III2IV1, thus affecting the functionality of the respiratory chain.

Show MeSH