Fig 2: SCO1 and SCO2 redox state and copper binding are regulated by COX11, PET191, and COX19.Potential redox and copper (Cu) binding states of SCO1 (A) or SCO2 (F). B, D, G, I Reverse SCO1 or SCO2 thiol trapping in the indicated cell lines. C, E, H, J The graphs show the quantification of six (for SCO1) or five (for SCO2) independent experiments as in B, D, G, I, respectively. The bars represent the mean ± SD. Two-sided unpaired t-test, ****p < 0.0001. ns, no significant. Figures B, D, G, I are representative of six (B, D) or five (G, I) independent repetitions with similar results. Source data for B–D and H–J are provided as a Source Data file.

Fig 3: CcO subunits and metallochaperones undergo stable and transient interactions to drive and coordinate metal center assembly.A Co-immunoprecipitation (co-IP) of endogenous COX11 with newly synthesized mitochondrial polypeptides, identified in the right-hand side. The graph shows the densitometry (average ± SD) of three independent experiments. Black dots represent individual data points. Two-sided unpaired t-test, ****p < 0.0001. The ratio “a-COX11 IP signal vs. control background signal” for two parameters, COX1/ATP6 and COX2/ATP6, is presented. B Co-IP of COX11-FLAG with CcO subunits and assembly factors in the presence or absence of the crosslinker DSP. Ex, extract; Un, unbound; IP, immunoprecipitate. C Stable interactions among CcO assembly factors determined by IP followed by either immunoblotting (WB) or mass spectrometry (MS). D Transient interactions among CcO assembly factors determined by IP in the presence of the crosslinker DSP (dithiobis(succinimidyl propionate)) or the vehicle DMSO (dimethyl sulfoxide) followed by immunoblotting. The heat maps show the average quantification of three independent experiments. Two-sided unpaired t-test. *p < 0.05. Figures A, B are representative of three independent repetitions with similar results. Source data for A, B, D are provided as a Source Data file.

Fig 4: COX11 redox state and copper binding are regulated by PET191 and COX19.A Reverse thiol trapping approach to detect the native cysteine residues inaccessible to a cell-permeable alkylating compound (2-iodoacetamide, IAA) that upon denaturation with SDS (sodium dodecyl sulfate) and full reduction with TCEP (tris(2-carboxyethyl)phosphine) are bound to AMS (4-acetamido-4’-maleimidylstilbene-2,2’- disulfonic acid), which adds ~540 Da per thiol. Three potential scenarios are depicted to account for copper (Cu)-bound COX11 dimer and oxidized or reduced COX11 monomers. B–D Reverse COX11 thiol trapping in the indicated cell lines. The bar graphs represent the average ± SD of four independent experiments. Two-sided unpaired t-test, ****p < 0.0001. Figures B–D are representative of three independent repetitions with similar results. Source data for B–D are provided as a Source Data file.

Fig 5: COX11- and PET191-containing metallochaperone modules coordinate copper and heme center assembly to minimize accumulation of pro-oxidant deleterious CcO assembly intermediates.A ROS generation in the indicated cell lines measured using superoxide-sensitive dyes dihydroethidium (DHE) and MitoSOX and the H2O2-sensitive dichlorodihydrofluorescein diacetate (CM-H2DCFDA). The bar graphs are quantification (mean ± SD) of three independent experiments. Black dots represent individual data points. Two-sided unpaired t-test, ****p < 0.0001. B H2O2 sensitivity measured as the release of lactate dehydrogenase (LDH) to the growth medium. In the bottom graph, the inset shows immunoblot analysis of COX10 steady-state levels in WT and PET191-KO HEK293T cells following 10 days of treatment with siRNA-COX10 or non-targeting (NT) control. ACTIN was used as the loading control. The bar graphs are quantification (mean ± SD) of nine independent experiments. Black dots represent individual data points. Two-sided unpaired t-test, ****p < 0.0001. Black stars are for comparisons with WT, and red starts for comparison with PET191-KO. Immunoblots in this panel are representative of three independent repetitions with similar results. Source data for the immunoblots are provided as a Source Data file. C Experimental workflow for copper measurements. Mitochondria isolated from WT or KO cell lines expressing FLAG-tagged COX11 or PET191 were solubilized in native conditions (see details in the Methods section), then the extracts used for FLAG-immunoprecipitation (IP), the IPed proteins eluted, and analyzed their metal content by inductively coupled plasma MS (ICP-MS). The panel was created with Biorender.com. D Copper content in COX11- and PET191-containing metallochaperone modules measured by ICP-MS. Mitochondria were purified from COX11-KO or PET191-KO cells expressing the indicated FLAG-tagged proteins. The proteins were immunoprecipitated using anti-FLAG conjugated beads, and their metal content was assessed by ICP-MS. The data is expressed as total Cu normalized by [34S]. The bar graphs are quantification (mean ± SD) of three (COX11-KO + COX11-C219C), six (COX11-KO + COX11 and PET191-KO + PET191 or + PET191-C30A,C41A) or nine (WT, COX11-KO + PET191 or + PET191-C30A,C41A) independent samples. Black dots represent individual data points. Two-sided unpaired t-test, ****p < 0.0001. Source data for A, B, D are provided as a Source Data file.