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The Function of the Iron–Sulfur Cluster Assembly Machinery is involved in Electron Transport Chain Activity and Oxidative Stress in Saccharomyces cerevisiae
Author(s) -
GOMEZGALLARDO MAURICIO,
SANCHEZBRIONES LUIS A,
DIAZPEREZ ALMA L,
LOPEZALVAREZ ARNOLDO,
CORTESROJO CHRISTIAN,
AGUILARMENDEZ OSVALDO,
SAAVEDRAMOLINA ALFREDO,
CAMPOSGARCIA JESUS
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.863.1
Subject(s) - mitochondrion , oxidative stress , respiratory chain , reactive oxygen species , electron transport chain , oxidative phosphorylation , saccharomyces cerevisiae , chemistry , biochemistry , intracellular , biophysics , iron–sulfur cluster , menadione , microbiology and biotechnology , biology , yeast , enzyme
For their function, many proteins depend on cofactor binding to specific sequence motifs. Fe‐S clusters are conserved and versatile inorganic cofactors that participate in a variety of processes and functions. In eukaryotes, biogenesis and recycling of Fe‐S clusters play important roles in the mechanisms of iron homeostasis involved in mitochondrial function. These Fe‐S clusters are assembled into apoproteins by the iron–sulfur cluster machinery (ISC). The present study aimed to determine the effects of mutations in the ISC genes on mitochondrial functionality and iron homeostasis under oxidative stress in S. cerevisiae BY4741 and its KanMX4 interruption ISC genes mutants: ssq1 Δ, grx5 Δ and isa1 Δ. Intracellular ROS, Fe 2+ and mitochondrial membrane potential in yeast cultures were determined using oxidant‐sensitive, cell‐permeant fluorescent probes (DHE, PGFL and Rho123). Fluorescence was quantified by flow cytometry, viewed and colocalized by confocal microscopy. Mitochondria were isolated and permeabilized for spectrophotometric determination of respiratory complexes activities. Oxygen consumption rate (OCR) was measured with a Clark‐type oxygen electrode coupled to a biological oxygen monitor. Electron transport chain (ETC) supercomplexes were identified by BN‐PAGE and Raman spectroscopy was performed to detect mitochondrial Fe‐S content. The results indicate that ROS generation caused by oxidizing agents like menadione is increased by ISC system dysfunction, due to loss of iron homeostasis (i.e. augmented free Fe 2+ ), suggesting that ROS and Fe 2+ from Fe‐S cluster proteins react creating a strong oxidative status in mitochondria. This was associated to an impairment on the activity of the complexes II, III and IV from the ETC in the ISC mutants. Indeed, increased oxygen generation instead consumption was observed in SSQ1 and ISA1 mutants suggesting a massive generation of ROS and its detoxification by superoxide dismutase and catalase. In mitochondria from SSQ1 and ISA1 mutants, the content of [Fe–S] centers was decreased along with III 2 IV 2 respiratory supercomplex formation, but not in the iron‐deficient ATX1 (cytosolic copper metallochaperone) and MRS4 (mitochondrial iron transporter) mutants, used as controls. In conclusion these results indicate that the ISC system is important in iron‐homeostasis, ROS stress and in the assembly of the supercomplexes III 2 IV 2 and III 2 IV 1 , thus affecting the functionality of the respiratory chain.