Mitochondrial Cytochrome c Oxidase Biogenesis Is Regulated by the Redox State of a Heme-Binding Translational Activator

Abstract

Aim: Mitochondrial cytochrome c oxidase (COX), the last enzyme of the respiratory chain, catalyzes the reduction of oxygen to water and therefore is essential for cell function and viability. COX is a multimeric complex, whose biogenesis is extensively regulated. One type of control targets cytochrome c oxidase subunit 1 (Cox1), a key COX enzymatic core subunit translated on mitochondrial ribosomes. In Saccharomyces cerevisiae, Cox1 synthesis and COX assembly are coordinated through a negative feedback regulatory loop. This coordination is mediated by Mss51, a heme-sensing COX1 mRNA-specific processing factor and translational activator that is also a Cox1 chaperone. In this study, we investigated whether Mss51 hemylation and Mss51-mediated Cox1 synthesis are both modulated by the reduction-oxidation (redox) environment.

Results: We report that Cox1 synthesis is attenuated under oxidative stress conditions and have identified one of the underlying mechanisms. We show that in vitro and in vivo exposure to hydrogen peroxide induces the formation of a disulfide bond in Mss51 involving CPX motif heme-coordinating cysteines. Mss51 oxidation results in a heme ligand switch, thereby lowering heme-binding affinity and promoting its release. We demonstrate that in addition to affecting Mss51-dependent heme sensing, oxidative stress compromises Mss51 roles in COX1 mRNA processing and translation.

Innovation: H2O2-induced downregulation of mitochondrial translation has so far not been reported. We show that high H2O2 concentrations induce a global attenuation effect, but milder concentrations specifically affect COX1 mRNA processing and translation in an Mss51-dependent manner.

Conclusion: The redox environment modulates Mss51 functions, which are essential for regulation of COX biogenesis and aerobic energy production.

FIG. 1.

H₂O₂-induced oxidative stress attenuates Cox1 synthesis in vivo. (A) In vivo mitochondrial protein synthesis (pulses of 15 min) in the indicated strains. Yeast cultures were grown to early exponential phase (OD₆₀₀ = 1) and subjected to treatment with different concentrations of H₂O₂ for 2 h at 30°C. (B) Immunoblot analysis performed using whole yeast cell lysates and an anti-Mss51 antibody. Bar graphs represent mean ± SD of percent of untreated control in three independent experiments. Cox1, cytochrome c oxidase subunit 1; OD, optical density; SD, standard deviation.

FIG. 2.

The redox state of recombinant Mss51-TF determines hemin coordination. (A) Mss51-TF redox state in vitro by thiol trapping. Purified recombinant Mss51-TF aliquots were left untreated, reduced (10 mM dithiothreitol), or oxidized (1 mM 4-DPS) and subsequently exposed to Mal-PEG5000. Proteins were resolved in SDS-PAGE under nonreducing conditions and Mss51 was detected by immunoblotting. (B) Difference spectroscopy titration of hemin binding to reduced or oxidized Mss51-TF performed with increasing concentrations of Fe³⁺ hemin. The titration curves (left panel) were generated from fits to equation, , describing a single binding site using GraphPad Prism software. The right panel displays the overlay of titration curves showing peaks, maxima, after binding 2 μM hemin. (C) Difference absorption spectra of hemin-bound native Mss51-TF treated with increasing concentrations of 4-DPS (left panel) and H₂O₂ (right panel), as shown. The results were obtained and analyzed as explained in (B). (D) Fluorescence emission spectra of reduced and oxidized Mss51-TF upon binding to hemin. Spectra were recorded at 25°C in 50 mM Tris-HCl pH 8.0 (excitation at 295 nm). The fluorescence maximum of reduced Mss51-TF was arbitrarily set to 1 fluorescence unit. For each condition, plots from three independent samples are presented. 4-DPS, aldrithiol-4; redox, reduction–oxidation; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

FIG. 3.

Exposure to H₂O₂ induces Mss51 oxidation in organello. (A) Mitochondria with intact outer membranes purified in the absence of reducing agents were treated with the indicated H₂O₂ concentrations and subsequently extracted with 100 mM Hepes pH 7.4 and 0.5% SDS with or without reducing agent, TCEP (tris-[2-carboxyethyl] phosphine, hydrochloride). Oxidation of Mss51 thiols was visualized by addition of AMS and immunoblotting. (B) Oxidation of mutant forms of Mss51-carrying serine substitutions for one or two cysteines. H₂O₂ treatment was followed by a thiol-trapping assay as in (A).

FIG. 4.

Exposure to H₂O₂ attenuates Cox1 synthesis in strains expressing mss51 cysteine mutants. (A) In vivo mitochondrial protein synthesis in the indicated strains grown to early exponential phase and subjected to treatment with different concentrations of H₂O₂ for 2 h at 30°C. The left bottom panel shows an immunoblot analysis performed using whole yeast cell lysates, an anti-Mss51 antibody, and an anti-Porin Ab as a loading control. (B) Growth test using serial dilutions of the indicated strains in complete media containing fermentable (glucose, YPD) or nonfermentable (ethanol-glycerol, YPEG) carbon sources. The plates were incubated at 30°C and the pictures taken after 2 days of growth. (C) In vivo mitochondrial proteins using untreated cultures of the indicated strains. Signals were quantified as in Figure 1.

FIG. 5.

Interventions that bypass COX1 mRNA translational downregulation do not bypass the H₂O₂-induced decrease in Cox1 synthesis. (A–F) In vivo mitochondrial protein synthesis performed as in Figure 4A using the indicated strains. (A, B) The right panel shows an immunoblot analysis of whole yeast cell lysates for Mss51 and Porin as a loading control. (C) The pulse was followed by a chase in the presence of cold methionine and puromycin to inhibit protein synthesis. Signals were quantified as in Figure 1.

FIG. 6.

Mss51 can be oxidized in vivo by H₂O₂. (A) In vivo mitochondrial protein synthesis performed as in Figure 4A using the indicated strains. (B) Reverse thiol-trapping assay to test the native Mss51 redox state in mitochondria isolated from the cultures used in (A). Numbers 1, 2, and 3 indicate the order in which each sample was treated with the thiol-binding compounds, IAM (iodoacetamide) and AMS (4-acetamido-4′maleimidylstilbene-2,2′-disulfonic acid), and the reducing agent, TCEP. Full reduction of Mss51 with TCEP (1) was tested by reducing the protein first, and then adding excess IAM (2), followed by AMS addition (3). Maximum shift of reduced Mss51 was determined by reducing the sample during extraction with TCEP (1), quickly followed by treatment with AMS (2). (C) Sucrose gradient sedimentation analysis of Mss51 high-molecular-mass complexes extracted in native conditions from treated and untreated mitochondria used in (B). The gradients were calibrated with the standards, hemoglobin (Hb, ∼67 kDa) and LDH (∼130 kDa). Fractions were resolved using SDS-PAGE and analyzed by immunoblotting for Mss51. Hb, hemoglobin; LDH, lactic dehydrogenase.

FIG. 7.

The oxidative stress-induced COX1 mRNA translational defect cannot be solely attributed to loss of heme binding to Mss51. (A) Redox state of Mss51 in mitochondria purified from the indicated strains and analyzed by thiol trapping as in Figure 6B. The Δhem1 mutation was bypassed by growing the cells in media containing 5-aminolevulinic acid (ALA) or expressed by growing cells in media lacking ALA, but supplemented with Tween 20 and ergosterol (TE) for 16 h before mitochondrial purification. (B) In vivo mitochondrial protein synthesis following ³⁵S-methionine incorporation as in Figure 4, using Δmss51 cells expressing from an integrative plasmid either wild-type MSS51 or the point mutant mss51^F199I. Signals were quantified as in Figure 1. (C) Native redox state of Mss51 in mitochondria isolated from the indicated strains analyzed by reverse thiol trapping as in Figure 6B. (D) Native distribution of Mss51 in sucrose gradients analyzed as in Figure 6C.

FIG. 8.

H₂O₂-induced oxidative stress negatively affects Mss51-dependent processing of intron-containing COX1 mRNA. (A, B) Yeast cultures of intron-containing (I⁺) and intronless (I⁰) strains were grown to early exponential phase and treated with the indicated concentrations of H₂O₂ for 2 h at 30°C. Treatment was followed by in vivo mitochondrial protein synthesis assay as in Figure 3 and signals quantified as in Figure 1. (C) Redox state of Mss51 in organello after treatment with H₂O₂. Intact mitochondria purified from strains carrying COX1 with (I⁺) or without introns (I⁰) were treated under isotonic conditions with the indicated H₂O₂ concentrations, and then subjected to a thiol-trapping assay as in Figure 3. (D) Native distribution in sucrose gradients of Mss51 from the same strains than in (C) analyzed as in Figure 6C. (E) Steady-state levels of total, processed, and unprocessed COX1 mRNA in the indicated strains measured by quantitative PCR. Levels of total COX1 mRNA were measured using primers within COX1 exon 4. Splicing of COX1 intron 1 was monitored by amplifying COX1 mRNA with primers designed either within intron 1 or at the end of exon 1 and the beginning of exon 2. The rho zero (ρ⁰) strain, devoid of mitochondrial DNA, was used as a negative control. Expression levels of COX1 mRNA were normalized by actin levels. Error bars represent the mean ± SD of three independent repetitions. *p < 0.05, **p < 0.01.

FIG. 9.

Effect of H₂O₂-induced oxidative stress in Mss51 properties and functions. According to our model, oxidative stress induces the formation of a disulfide bond in Mss51, involving the two cysteines in the CPX heme-regulatory motifs, and a change in the conformation of the protein. At least one of the CPX cysteines coordinates heme B in a noncovalent manner. Mss51 oxidation results in heme ligand switch, which lowers heme-binding affinity and induces its release. Oxidation also provokes a change in Mss51 conformation. These changes markedly inhibit Mss51 functions in COX1 mRNA processing and translation and Cox1 assembly chaperoning. In this way, Mss51 acts as a redox sensor to discontinue Cox1 synthesis and COX assembly during potentially harmful oxidative stress conditions.