Dynamic assessment of the allocation of copper to cytochrome c oxidase using size-exclusion chromatography (SEC) combined with inductively coupled plasma mass spectrometry (ICP-MS).

Copper (Cu) is an essential trace element required for mitochondrial respiration via its incorporation into cytochrome c oxidase (CuCOX), the terminal enzyme of the electron transport chain. Here, we employed size-exclusion chromatography coupled with inductively coupled plasma mass spectrometry (SEC-ICP-MS), UV-Vis spectroscopy, and immunoblotting to identify and validate a high molecular weight Cu-containing peak in the SEC-ICP-MS chromatogram as representative of CuCOX activity. We demonstrate that this CuCOX peak is enhanced under metabolic conditions inducing oxidative phosphorylation, such as high Cu supplementation or galactose-containing media, and correlates with increased mitochondrial respiration. Using exogenous (63)Cu tracing, we characterized the time- and dose-dependent incorporation of newly acquired Cu into CuCOX under elevated Cu conditions in renal cancer cells, modeling advanced clear cell renal cell carcinoma (ccRCC). RNA interference experiments targeting key Cu transporters revealed that CuCOX formation is independent of the high-affinity Cu importer CTR1, but instead relies on alternative transporters, including DMT1, LAT1, and the mitochondrial carrier SLC25A3, with transporter contributions dynamically reshaped during chronic adaptation to high Cu availability. In contrast, under standard low-Cu conditions, CTR1 remains required for cellular Cu uptake and CuCOX metallation. Together, these findings define context-dependent Cu trafficking pathways in renal cancer and establish SEC-ICP-MS as a sensitive platform for assessing CuCOX metallation and mitochondrial metabolism, with potential applications in biomarker discovery and therapeutic targeting in RCC.