Mitochondrial COA7 is a heme-binding protein with disulfide reductase activity, which acts in the early stages of complex IV assembly

Abstract

Cytochrome c oxidase (COX) assembly factor 7 (COA7) is a metazoan-specific assembly factor, critical for the biogenesis of mitochondrial complex IV (cytochrome c oxidase). Although mutations in COA7 have been linked to complex IV assembly defects and neurological conditions such as peripheral neuropathy, ataxia, and leukoencephalopathy, the precise role COA7 plays in the biogenesis of complex IV is not known. Here, we show that loss of COA7 blocks complex IV assembly after the initial step where the COX1 module is built, progression from which requires the incorporation of copper and addition of the COX2 and COX3 modules. The crystal structure of COA7, determined to 2.4 Å resolution, reveals a banana-shaped molecule composed of five helix-turn-helix (α/α) repeats, tethered by disulfide bonds. COA7 interacts transiently with the copper metallochaperones SCO1 and SCO2 and catalyzes the reduction of disulfide bonds within these proteins, which are crucial for copper relay to COX2. COA7 binds heme with micromolar affinity, through axial ligation to the central iron atom by histidine and methionine residues. We therefore propose that COA7 is a heme-binding disulfide reductase for regenerating the copper relay system that underpins complex IV assembly.

Keywords: COA7; X-ray crystallography; cytochrome c oxidase; heme; mitochondria.

Fig. 1.

Loss of COA7 results in complex IV assembly defects. (A and B) Mitochondria isolated from control, COA7^KO-1, COA7^KO-2, and rescued COA7^KO-1 cells were solubilized in 1% digitonin and subjected to BN-PAGE and analyzed using antibodies against subunits in brackets. Coomassie brilliant blue (CBB)–stained membranes showing complex V (CV) are indicative of loading. (C) Cells were pulsed with [³⁵S]-Met for 2 h and chased for the indicated times. Isolated mitochondria were analyzed by SDS-PAGE and phosphorimaging. Immunoblotting (IB) with the complex II subunit SDHA was used as a loading control. (D) Volcano plot showing proteins with altered abundance in COA7^KO mitochondria. The SILAC log₂-transformed fold change (FC) ratios are indicated on the x-axis. Subunits of the different OXPHOS complexes are color-coded, and complex IV subunits are labeled. The horizontal line indicates P = 0.05, and vertical lines indicate a fold change of 1.5. Topographical heatmap of SILAC log₂-transformed fold change (FC) ratios mapped onto the structure of complex IV (PDB 5Z62) (31). Proteins encoded by mtDNA are shown as surface renders, while nuclear-encoded proteins are shown as cartoons. Blue intensity is relative to the decrease in protein abundance. n.d., not detected. (E) Venn diagram summarizing COA7^Flag-interacting proteins under native conditions or following chemical cross-linking of cells grown in Glu- or Gal-containing media. Complex IV subunits and assembly factors are in bold. N/A; not applicable.

Fig. 2.

Structure of COA7 and analyses of pathogenic variants. (A) Cartoon representation of the overall structure of COA7. Secondary structures are represented as cartoons with SEL1 repeats colored in gray, raspberry, cyan, teal, and green. The transparent molecular surface is represented in gray. The amino (N) and carboxy (C) termini are indicated. (B) Cysteine residues (labeled) involved in intramolecular disulfide bonds are shown as yellow spheres, and free cysteine residues are shown as salmon spheres. (C) The molecular surface of COA7 is colored according to the electrostatic potentials (red, negatively charged; blue, positively charged; white, uncharged). The Top images show the side view of the COA7 structure. The Left and Right Bottom images show concave and convex views, respectively. The amino (N) and carboxy (C) termini are indicated. (D) Mitochondria isolated from control, COA7^KO cells, and COA7^KO cells expressing wildtype (WT) COA7^Flag and variants in the absence or presence of doxycycline (DOX) as indicated were subjected to SDS-PAGE and immunoblotting with antibodies as shown. ATP5A serves as a loading control. (E) Patient mutations are mapped onto the structure of COA7 and shown as pink sticks and hydrogen bonds between residues (labeled) shown as dashed lines. Inset: Closer view of the interactions formed by residues His112 and Tyr137. The amino (N) and carboxy (C) termini are indicated.

Fig. 3.

Heme binding to COA7. (A) Difference absorption spectra of COA7 (1 μM) with increasing concentrations of Fe^(III)-PPIX (from 0 to 10 μM) as indicated. (B) The binding curve was fitted to the data from A using an equation describing a single binding site (Y = Bmax * X/K_D + X) with GraphPad Prism. (C) UV-Vis absorption spectra of COA7 in the presence of one molar equivalent of Fe^(III)-PPIX (black); Fe^(II)-PPIX (red), and Zn^(II)-PPIX (dotted, black).

Fig. 4.

COA7 functions as a disulfide reductase in complex IV assembly. (A) Schematic of the heme biosynthetic pathway. Succinylacetone (SA) inhibits the second enzyme in the pathway, 5-aminolevulinic acid dehydratase (ALAD). 5-aminolevulinic acid synthase 1 (ALAS1), 5-aminolevulinic acid (5-ALA), porphobilinogen (PBG), coproporphyrinogen oxidase (CPOX), ferrochelatase (FECH). (B) The relative abundance of COX1 in mitochondria isolated from the cells indicated were quantified by densitometry. n = 4, mean ± SEM, ordinary one-way ANOVA, using Tukey’s multiple comparisons test, ****P < 0.0001; n.s., not significant. (C) Analysis of the reduction of a single disulfide bond in SCO1 by Fe^(II)-heme-COA7 via electrospray ionization mass spectrometry. The Top spectrum shows a deconvoluted experimental molecular weight of oxidized SCO1 (S-S) following iodoacetamide labeling (19,766.52 Da). The Bottom spectrum shows a deconvoluted experimental molecular weight of reduced SCO1 following reduction by Fe^(II)-heme-COA7 and iodoacetamide labeling (S-IAA, 19,881.15 Da). (D) Analysis of the reduction of a single disulfide in SCO2 by Fe^(II)-heme-COA7 via electrospray ionization mass spectrometry. The Top spectrum shows a deconvoluted experimental molecular weight of oxidized SCO2 (S-S) following iodoacetamide labeling (S-IAA, 19,543.48 Da). The Bottom spectrum shows a deconvoluted experimental molecular weight of reduced SCO2 following reduction by Fe^(II)-heme-COA7 and iodoacetamide labeling (19,660.03 Da). (E) Mitochondria isolated from control, COA7^KO, COA7^KO+COA7^Flag (+COA7^Flag), and HIGD2A^KO cells were subjected to redox analysis by treatment with DTT, iodoacetamide (IAA), and AMS and analyzed by SDS-PAGE and immunoblotting (IB) with SCO1 and SCO2 antibodies as indicated. RED, reduced SCO1/SCO2; OX, oxidized SCO1/SCO2.