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.