To minimize copper (Cu) toxicity, organisms have evolved Cu transport pathways involving soluble metallochaperones that bind, transport, and deliver Cu+ to specific partner proteins, such as Cu-ATPases. The human Cu chaperone, Atox1, delivers Cu to the metal-binding domains of Menkes (MNK) and Wilson (WND) disease proteins that are Cu-ATPases in the Golgi network that transfer Cu to cuproenzymes (e.g., ceruloplasmin) that traverse the Golgi lumen. The metal binding motif, MetX1CysXXCys, and the ferredoxin-like fold appear conserved in both cytoplasmic Cu chaperones and the cytoplasmic metal-binding domains of the target Cu-ATPases from different organisms. The work reported here provides a basic understanding of in vitro holo- and apo-protein stability, Cu-dissociation mechanisms, and donor-acceptor interactions of key copper transport chaperones. Studies were conducted on purified protein variants using circular dichroism, fluorescence, and absorbance methods in equilibrium and time-resolved modes. We developed a kinetic assay to determine the Cu-dissociation mechanism of these proteins and a near-UV CD method for monitoring interactions between Atox1 and WND domains to complement NMR measurements and computer simulations. Despite the conservation of the overall structural fold, the chaperones Atox1 and its bacterial homolog, CopZ, and the metal-binding domains of WND, W2 and W4, have variable chemical and thermal stability in vitro. The role of residues proximal to the metal-binding site was determined using Atox1 as a prototypical Cu chaperone. Met10 is essential for structural stability of Atox1. Thr11 (position X1) seems to be conserved, not for integrity of protein structure, but for facilitating metal exchange between Atox1 and a receptor domain. The structural proximity of the charged side-chain of Lys60 neutralizes the Cu-thiolate center in Atox1. Replacement of Lys60 with an Ala or Tyr results in a higher rate and extent of loss of the metal to small molecule chelator, BCA, than those for wtAtox1. Lys60 also provides electrostatic interactions crucial for Atox1 interaction with W4. Thus, each proximal residue contributes to fine-tuning copper binding and its release mechanism to both the non-physiological Cu chelator, BCA, and the physiological acceptor of the WND protein, W4. Our new kinetic and spectral assays provide a comprehensive in vitro experimental platform for more advanced future mechanistic and kinetic studies.