The superior Cu(ii) affinity of human copper transporter 1 (hCtr1) drives copper acquisition from human serum albumin (HSA).

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 catabolism of β-amyloid (Aβ) is carried out by numerous endopeptidases including neprilysin, which hydrolyzes peptide bonds preceding positions 4, 10, and 12 to yield Aβ4–9 and a minor Aβ12–x species. Alternative processing of the amyloid precursor protein by β-secretase also generates the Aβ11–x species. All these peptides contain a Xxx-Yyy-His sequence, also known as an ATCUN or NTS motif, making them strong chelators of Cu­(II) ions. We synthesized the corresponding peptides, Phe-Arg-His-Asp-Ser-Gly-OH (Aβ4–9), Glu-Val-His-His-Gln-Lys-am (Aβ11–16), Val-His-His-Gln-Lys-am (Aβ12–16), and pGlu-Val-His-His-Gln-Lys-am (pAβ11–16), and investigated their Cu­(II) binding properties using potentiometry, and UV–vis, circular dichroism, and electron paramagnetic resonance spectroscopies. We found that the three peptides with unmodified N-termini formed square-planar Cu­(II) complexes at pH 7.4 with analogous geometries but significantly varied K d values of 6.6 fM (Aβ4–9), 9.5 fM (Aβ12–16), and 1.8 pM (Aβ11–16). Cyclization of the N-terminal Glu11 residue to the pyroglutamate species pAβ11–16 dramatically reduced the affinity (5.8 nM). The Cu­(II) affinities of Aβ4–9 and Aβ12–16 are the highest among the Cu­(II) complexes of Aβ peptides. Using fluorescence spectroscopy, we demonstrated that the Cu­(II) exchange between the Phe-Arg-His and Val-His-His motifs is very slow, on the order of days. These results are discussed in terms of the relevance of Aβ4–9, a major Cu­(II) binding Aβ fragment generated by neprilysin, as a possible Cu­(II) carrier in the brain.

The apparent affinity of human serum albumin (HSA) for divalent copper has long been the subject of great interest, due to its presumed role as the major Cu2+‐binding ligand in blood and cerebrospinal fluid. Using a combination of electronic absorption, circular dichroism and room‐temperature electron paramagnetic resonance spectroscopies, together with potentiometric titrations, we competed the tripeptide GGH against HSA to reveal a conditional binding constant of log cKCuCu(HSA) =13.02±0.05 at pH 7.4. This rigorously determined value of the Cu2+ affinity has important implications for understanding the extracellular distribution of copper.

The Aβ 5– x peptides ( x = 38, 40, 42) are minor Aβ species in normal brains but elevated upon the application of inhibitors of Aβ processing enzymes. They are interesting from the point of view of coordination chemistry for the presence of an Arg-His metal binding sequence at their N-terminus capable of forming a 3-nitrogen (3N) three-coordinate chelate system. Similar sequences in other bioactive peptides were shown to bind Cu(II) ions in biological systems. Therefore, we investigated Cu(II) complex formation and reactivity of a series of truncated Aβ 5– x peptide models comprising the metal binding site: Aβ 5–9 , Aβ 5–12 , Aβ 5–12 Y10F, and Aβ 5–16 . Using CD and UV–vis spectroscopies and potentiometry, we found that all peptides coordinated the Cu(II) ion with substantial affinities higher than 3 × 10 12 M –1 at pH 7.4 for Aβ 5–9 and Aβ 5–12 . This affinity was elevated 3-fold in Aβ 5–16 by the formation of the internal macrochelate with the fourth coordination site occupied by the imidazole nitrogen of the His13 or His14 residue. A much higher boost of affinity could be achieved in Aβ 5–9 and Aβ 5–12 by adding appropriate amounts of the external imidazole ligand. The 3N Cu-Aβ 5– x complexes could be irreversibly reduced to Cu(I) at about −0.6 V vs Ag/AgCl and oxidized to Cu(III) at about 1.2 V vs Ag/AgCl. The internal or external imidazole coordination to the 3N core resulted in a slight destabilization of the Cu(I) state and stabilization of the Cu(III) state. Taken together these results indicate that Aβ 5– x peptides, which bind Cu(II) ions much more strongly than Aβ 1– x peptides and only slightly weaker than Aβ 4– x peptides could interfere with Cu(II) handling by these peptides, adding to copper dyshomeostasis in Alzheimer brains.