Designing small peptides that are capable of binding Cu2+ ions mainly through the side-chain functionalities is a hard task because the amide nitrogen atoms strongly compete for Cu2+ ion coordination. However, the design of such peptides is important for obtaining biomimetic small systems of metalloenyzmes as well as for the development of artificial systems. With this in mind, a cyclic decapeptide, C-Asp, which contained three His residues and one Asp residue, and its linear derivative, O-Asp, were synthesized. The C-Asp peptide has two ProGly -turn-inducer units and, as a result of cyclization, and as shown by CD spectroscopy, its backbone is constrained into a more defined conformation than O-Asp, which is linear and contains a single ProGly unit. A detailed potentiometric, mass spectrometric, and spectroscopic study (UV/Vis, CD, and EPR spectroscopy) showed that at a 1:1 Cu2+/peptide ratio, both peptides formed a major [CuHL]2+ species in the pH range 5.07.5 (C-Asp) and 5.57.0 (O-Asp). The corrected stability constants of the protonated species (logK*CuH(OAsp)=9.28 and logK*CuH(CAsp)=10.79) indicate that the cyclic peptide binds Cu2+ ions with higher affinity. In addition, the calculated value of Keff shows that this higher affinity for Cu2+ ions prevails at all pH values, not only for a 1:1 ratio but even for a 2:1 ratio. The spectroscopic data of both [CuHL]2+ species are consistent with the exclusive coordination of Cu2+ ions by the side-chain functionalities of the three His residues and the Asp residue in a square-planar or square-pyramidal geometry. Nonetheless, although these data show that, upon metal coordination, both peptides adopt a similar fold, the larger conformational constraints that are present in the cyclic scaffold results in different behaviour for both [CuHL]2+ species. CD and NMR analysis revealed the formation of a more rigid structure and a slower Cu2+-exchange rate for [CuH(C-Asp)]2+ compared to [CuH(O-Asp]2+. This detailed comparative study shows that cyclization has a remarkable effect on the Cu2+-coordination properties of the C-Asp peptide, which binds Cu2+ ions with higher affinity at all pH values, stabilizes the [CuHL]2+ species in a wider pH range, and has a slower Cu2+-exchange rate compared to O-Asp.