# Intermediate CuIIATCUN Species Reacts With Biomolecules Within Time Windows of Biological Processes

## TL;DR

A copper-binding motif in proteins forms a reactive intermediate that may play a role in biological copper processes.

## Contribution

The discovery that a transient CuII intermediate formed by the ATCUN motif is reactive and redox-active.

## Key findings

- The 2N intermediate CuII species formed with a model ATCUN peptide is labile and exchanges copper with histidine.
- The intermediate is rapidly reduced to CuI by glutathione, unlike the final 4N complex.
- These findings suggest the intermediate's physiological relevance in copper redox and exchange reactions.

## Abstract

The ATCUN motif is the N‐terminal peptide/protein sequence Xaa‐Zaa‐His, bearing the N‐terminal amine and Zaa ≠ Pro, which effectively binds some transition metal ions including CuII in a square‐planar geometry. This motif has attracted much attention, due to its distinctive copper binding properties and abundance in human proteome. However, the inertness and poor reactivity of CuIIATCUN complexes appear at odds with the biological reality of dynamically changing copper balance, which is maintained through redox and trans‐chelation reactions. We have demonstrated a multi‐step character of Cu2+ binding to ATCUN sequences, with partially coordinated intermediate species present within time windows spanning from hundreds of milliseconds to seconds. Based on indirect evidence and chemical analogy we previously proposed that these transient species could serve as reactive intermediates in biological copper redox and exchange reactions. Here we used double mixing stopped‐flow experiments to address this issue directly for a model tetrapeptide (DAHK) representing the ATCUN motif at the N‐terminus of Human Serum Albumin. We found that, unlike the final 4‐nitrogen coordinated (4N) square‐planar complex, the transient 2N intermediate readily exchanged CuII with histidine and was rapidly reduced to CuI by glutathione. These results pave way to re‐evaluation of physiological roles of ATCUN motifs.

We have demonstrated that a partially coordinated intermediate species formed in a multistep reaction of the Cu(II) ion with a model peptide representing the N‐terminus of Human Serum Albumin exhibits remarkable lability and redox‐activity, which strongly supports its physiological relevance.