# 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)

## TL;DR

The study uses a new method to track how copper is used in a key mitochondrial enzyme and finds that copper transporters change depending on copper availability in kidney cancer cells.

## Contribution

A novel SEC-ICP-MS method is introduced to dynamically assess copper allocation to cytochrome c oxidase in renal cancer cells.

## Key findings

- A high molecular weight copper-containing peak in SEC-ICP-MS correlates with cytochrome c oxidase activity under oxidative phosphorylation conditions.
- Copper incorporation into cytochrome c oxidase in renal cancer cells is time- and dose-dependent under high copper conditions.
- Copper transporter contributions to cytochrome c oxidase formation shift dynamically with copper availability.

## Abstract

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 63Cu 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.