Cu(II) Binding to the Peptide Ala-His-His, a Chimera of the Canonical Cu(II)-Binding Motifs Xxx-His and Xxx-Zzz-His

Abstract

Peptides and proteins with the N-terminal motifs NH-Xxx-His and NH-Xxx-Zzz-His form well-established Cu(II) complexes. The canonical peptides are Gly-His-Lys and Asp-Ala-His-Lys (from the wound healing factor and human serum albumin, respectively). Cu(II) is bound to NH-Xxx-His via three nitrogens from the peptide and an external ligand in the equatorial plane (called 3N form here). In contrast, Cu(II) is bound to NH-Xxx-Zzz-His via four nitrogens from the peptide in the equatorial plane (called 4N form here). These two motifs are not mutually exclusive, as the peptides with the sequence NH-Xxx-His-His contain both of them. However, this chimera has never been fully explored. In this work, we use a multispectroscopic approach to analyze the Cu(II) binding to the chimeric peptide Ala-His-His (AHH). AHH is capable of forming the 3N- and 4N-type complexes in a pH dependent manner. The 3N form predominates at pH ∼ 4-6.5 and the 4N form at ∼ pH 6.5-10. NMR experiments showed that at pH 8.5, where Cu(II) is almost exclusively bound in the 4N form, the Cu(II)-exchange between AHH or the amidated AHH-NH is fast, in comparison to the nonchimeric 4N form (AAH). Together, the results show that the chimeric AHH can access both Cu(II) coordination types, that minor changes in the second (or further) coordination sphere can impact considerably the equilibrium between the forms, and that Cu kinetic exchange is fast even when Cu-AHH is mainly in the 4N form.

Triggering Cu-coordination change in Cu(ii)-Ala-His-His by external ligands

“…1; Table S1 & 2 for EPR parameters). 15 The addition of Im (Fig. 1b, orange) yielded a spectrum with g// = 2.21(1) and A// 596 MHz, very similar to that of the canonical Cu(GHK) ternary complex with Im 11 , matching the formation of the 3N+N Im complex (for AH see Fig.…”

“…This includes their spectroscopic properties, correlated with one different donor atom. 14,15 The time scales of Cu(II) self-exchange reactions are dramatically different: they are slow for XZH (minutes) but fast for XH (< seconds). 16 With respect to redox properties, Cu(II) coordinated to XZH can be oxidized to Cu(III) while Cu(II) in a Cu(XH) complex can be reduced to Cu(I) in the [-1.0,+1.0 V/vs SCE range].…”

“…17 We previously investigated the Cu(II) complexes of Ala-His-His-OH (AHH) and Ala-His-His-NH2 (AHH-am) peptides, chimeras of these two motifs, and demonstrated that they actuate the 3N and the 4N binding modes reversibly as a function of pH. 15 The same modes were also described beforehand by Jackson et al for the peptide sarcosyl-His-His, which has a secondary amine instead of the primary one at the N-terminus. 18 Taking advantage of the labile equatorial site in the 3N coordination mode, the present work explores the ability of external, nitrogen bearing ligands L to switch between the 3N+L and 4N binding modes in the Cu(AHH) system for L including imidazole and azide, seeking their effect on Cu(II) reactivity in the biotechnological and biochemical context.…”

N‐Terminal Cu‐Binding Motifs (Xxx‐Zzz‐His, Xxx‐His) and Their Derivatives: Chemistry, Biology and Medicinal Applications

“…Cu‐XZH could not be electrochemically reduced down to −1.0 V vs. NHE . In contrast, Cu II ‐XH complexes cannot be oxidized up to 1.2 V vs. NHE, but can be reduced at a cathodic potential of about −0.2 V vs. NHE . The reduction is electrochemically irreversible leading to two anodic peaks on the reverse scan at about 0.1 and 0.3 V. vs. NHE, corresponding to oxidation of uncoordinated Cu I and of Cu I bound to two His residues, respectively.…”

“…[2] In contrast, Cu II -XH complexes cannotb eo xidized up to 1.2V vs. NHE, but can be reduced at ac athodic potential of about À0.2 Vv s. NHE. [2,40] The reduction is electrochemically irreversible leadingt ot wo anodic peaks on ther everses can at about 0.1 and 0.3 V. vs. NHE, corresponding to oxidation of uncoordinated Cu I and of Cu I boundt ot wo His residues, respectively.I nt he presence of more than one His or with an exogenous imidazole source,t he first cathodic peak is no more observed. The electrochemical properties of both systemsa re summarized in Figure3,i n which they are compared with the chemical redoxp roperties of both system.…”