Human carbonic anhydrase (CA) is a well-studied, robust, mononuclear Zn-containing metalloprotein that serves as an excellent biological ligand system to study the thermodynamics associated with metal ion coordination chemistry in aqueous solution. The apo form of human carbonic anhydrase II (CA) binds 2 equiv of copper(II) with high affinity. The Cu2+ ions bind independently forming two noncoupled type II copper centers in CA (Cu-A and Cu-B). However, the location and coordination mode of the Cu-A site in solution is unclear, compared to the Cu-B site that has been well-characterized. Using paramagnetic NMR techniques and X-ray absorption spectroscopy we identified an N-terminal Cu2+ binding location and collected information on the coordination mode of the Cu-A site in CA, which is consistent with a four- to five-coordinate N-terminal Cu2+ binding site reminiscent to a number of N-terminal copper(II) binding sites including the copper(II)-amino terminal Cu2+ and Ni2+ and copper(II)-beta-amyloid complexes. Additionally, we report a more detailed analysis of the thermodynamics associated with copper(II) binding to CA. Although we are still unable to fully deconvolute Cu2+ binding data to the high-affinity Cu-A site, we derived pH- and buffer-independent values for the thermodynamics parameters K and Delta H associated with Cu2+ binding to the Cu-B site of CA to be 2 x 10(9) and -17.4 kcal/mol, respectively.