Intermediate Cu^IIATCUN Species Reacts With Biomolecules Within Time Windows of Biological Processes

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 Cu^II 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 Cu^IIATCUN 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 Cu^II with histidine and was rapidly reduced to Cu^I by glutathione. These results pave way to re-evaluation of physiological roles of ATCUN motifs.