Mitochondrial Cytochrome c Oxidase Defects Alter Cellular Homeostasis of Transition Metals

Abstract

The redox activity of cytochrome c oxidase (COX), the terminal oxidase of the mitochondrial respiratory chain (MRC), depends on the incorporation of iron and copper into its catalytic centers. Many mitochondrial proteins have specific roles for the synthesis and delivery of metal-containing cofactors during COX biogenesis. In addition, a large set of different factors possess other molecular functions as chaperones or translocators that are also necessary for the correct maturation of these complexes. Pathological variants in genes encoding structural MRC subunits and these different assembly factors produce respiratory chain deficiency and lead to mitochondrial disease. COX deficiency in Drosophila melanogaster, induced by downregulated expression of three different assembly factors and one structural subunit, resulted in decreased copper content in the mitochondria accompanied by different degrees of increase in the cytosol. The disturbances in metal homeostasis were not limited only to copper, as some changes in the levels of cytosolic and/or mitochondrial iron, manganase and, especially, zinc were observed in several of the COX-deficient groups. The altered copper and zinc handling in the COX defective models resulted in a transcriptional response decreasing the expression of copper transporters and increasing the expression of metallothioneins. We conclude that COX deficiency is generally responsible for an altered mitochondrial and cellular homeostasis of transition metals, with variations depending on the origin of COX assembly defect.

Keywords: copper; cytochrome c oxidase; iron; manganese; metal homeostasis; mitochondrial respiratory chain; zinc.

FIGURE 1

Extent of the COX deficiency in D. melanogaster KD models. (A) Spectrophotometric kinetic enzyme activity measurements of COX activity, normalized by the activity of citrate synthase (COX/CS) in mitochondrial fractions from control adults (solid blue bars), Coa8 KD adults (solid red bars), Coa3 KD adults (solid orange bars), control larvae (dotted blue bars), cype KD larvae (dotted purple bars) and Scox KD larvae (dotted green bars). The symbols represent the individual values of each replicate measurement, and the bars represent the mean ± SD. The statistical significance was calculated using Student’s t-test for the adult pairwise comparisons, and one-way ANOVA with Tukey’s multiple comparisons test for the three larvae groups (**p ≤ 0.01). (B) COX in gel activity assays performed in DDM-solubilized mitochondrial samples, separated through blue-native electrophoresis gels, from three independent replicates for each of the indicated experimental group.

FIGURE 2

Cellular copper and iron compartmentalization in D. melanogaster models of COX deficiency. (A) Copper and (B) iron content in parts per billion (ppb) and normalized by protein content (mg protein) in cytosolic and mitochondrial fractions from control adults (solid blue bars), Coa8 KD adults (solid red bars), Coa3 KD adults (solid orange bars), control larvae (dotted blue bars), cype KD larvae (dotted purple bars) and Scox KD larvae (dotted green bars). The symbols represent the individual values of each replicate measurement, and the bars represent the mean ± SD. The statistical significance was calculated using two-way ANOVA and Sidak’s multiple comparison tests (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).

FIGURE 3

Cellular manganese and zinc compartmentalization in D. melanogaster models of COX deficiency. (A) Manganese and (B) zinc content in parts per billion (ppb) and normalized by protein content (mg protein) in cytosolic and mitochondrial fractions from control adults (solid blue bars), Coa8 KD adults (solid red bars), Coa3 KD adults (solid orange bars), control larvae (dotted blue bars), cype KD larvae (dotted purple bars) and Scox KD larvae (dotted green bars). The symbols represent the individual values of each replicate measurement and the bars represent the mean ± SD. The statistical significance was calculated using two-way ANOVA and Sidak’s multiple comparison tests (**p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).

FIGURE 4

COX deficiency alters the expression of metal-responsive transcription factor 1 (MTF-1) target genes. Transcript levels measured by quantitative PCR of the copper transporters Ctr1A, Ctr1B, and the metallothioneins MtnA, MtnD in different genetic models of COX deficiency. Expression levels are normalized to the expression in the control strains (set to 1). Genotypes are: Coa8 KD (solid red bars), Coa3 KD (solid orange bars), cype KD (dotted purple bars) and Scox KD (dotted green bars). The bars represent the mean ± SD of n = 3 biological replicates, each measured in triplicate. One-way ANOVA and Dunett’s multiple comparison test of KD vs. control (*p ≤ 0.05, **p ≤ 0.01 ***p ≤ 0.001). Ad: Adults; Lv: Larvae.