Site-Specific Detection of Copper–Peptide Coordination in Solution Phase by Two-Dimensional Infrared Spectroscopy

Elucidating the molecular details of copper (Cu) coordination in peptides is essential for understanding its homeostasis in biological systems and developing functional biomimetic catalysts. Conventional techniques, such as EXAFS, EPR, and UV–vis spectroscopy, provide merely global information but fail to unambiguously identify the specific donor groups involved in metal binding in peptides. To overcome this limitation, we have developed a site-specific approach using two-dimensional infrared spectroscopy and have achieved site-specific resolution of Cu coordination in histidine-containing peptides in the solution phase. By employing a systematic series of natural and site-specific 13 C 15 N isotope-labeled oligopeptides, we established a set of IR markers that fingerprint individual donor groups, including backbone amides, His side chains, and peptide termini. Using these IR markers, we demonstrate that the amide I band serves as a sensitive probe for monitoring stepwise Cu(II) ligation processes and tracking monomer–dimer equilibrium. By isotopic labeling, we further identified the primary Cu binding site within a 16-mer histidine-rich peptide. The observed preferential Cu binding at the peptide N terminus underscores the potential of this approach for probing Cu coordination dynamics in diverse peptide and protein systems. The 2D IR methodology established herein provides a novel and general platform for probing metal–peptide interactions in the solution phase, offering a direct pathway to elucidate site-specific metal binding, resolve complex structural rearrangements, and capture transient intermediates in diverse peptide and protein systems.