The amino‐terminal copper and nickel/N‐terminal site (ATCUN/NTS) present in proteins and bioactive peptides exhibits high affinity towards Cu II ions and have been implicated in human copper physiology. Little is known, however, about the rate and exact mechanism of formation of such complexes. We used the stopped‐flow and microsecond freeze‐hyperquenching (MHQ) techniques supported by steady‐state spectroscopic and electrochemical data to demonstrate the formation of partially coordinated intermediate Cu II complexes formed by glycyl–glycyl–histidine (GGH) peptide, the simplest ATCUN/NTS model. One of these novel intermediates, characterized by two‐nitrogen coordination, t 1/2 ≈100 ms at pH 6.0 and the ability to maintain the Cu II /Cu I redox pair is the best candidate for the long‐sought reactive species in extracellular copper transport.

The Cu­(II) and Zn­(II) binding abilities of Gly-His-Thr-Asp-amide (GHTD-am), a tetrapeptide coreleased from the pancreas along with insulin, were studied using UV–vis and circular dichroism spectroscopies, potentiometry, and calorimetry. GHTD-am is a very strong Cu­(II) chelator, forming a three-nitrogen complex with a conditional affinity constant C K at pH 7.4 of 4.5 × 1012 M–1. The fourth coordination site can be occupied by a solvent molecule or a ternary ligand, such as imidazole, with C K on the order of several hundred reciprocal molar. The Zn­(II) binding ability of GHTD-am is relatively weak, with C K values at pH 7.4 of 3.0 × 104 and 2.0 × 103 M–1 for the first and second GHTD-am molecule coordinated, respectively. These results are discussed in light of the modes of interactions of Zn­(II) and Cu­(II) ions with insulin. A direct effect of GHTD-am on the Zn­(II) interactions with insulin is unlikely, but its Cu­(II) complex may have a biological relevance because of its high affinity and ability to form ternary complexes.

Multiple intermediates were found in Cu(ii) binding to Aβ4–16 before the formation of a tight complex.

The toolset of mass spectrometry (MS) is still expanding, and the number of metal ion complexes researched this way is growing. The Cu(II) ion forms particularly strong peptide complexes of biological interest which are frequent objects of MS studies, but quantitative aspects of some reported results are at odds with those of experiments performed in solution. Cu(II) complexes are usually characterized by fast ligand exchange rates, despite their high affinity, and we speculated that such kinetic lability could be responsible for the observed discrepancies. In order to resolve this issue, we selected peptides belonging to the ATCUN family characterized with high and thoroughly determined Cu(II) binding constants and re-estimated them using two ESI-MS techniques: standard conditions in combination with serial dilution experiments and very mild conditions for competition experiments. The sample acidification, which accompanies the electrospray formation, was simulated with the pH−jump stopped-flow technique. Our results indicate that ESI-MS should not be used for quantitative studies of Cu(II)−peptide complexes because the electrospray formation process compromises the entropic contribution to the complex stability, yielding underestimations of complex stability constants.

The aim of this perspective is to help revive studies of the kinetics of formation and exchange in Cu(ii)–peptide complexes. Such studies have the power to reveal novel reactive intermediates and set timeframes for actual physiological processes.