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 Cu(2+) ions bind independently forming two noncoupled type II copper centers in CA (CuA and CuB). However, the location and coordination mode of the CuA site in solution is unclear, compared to the CuB site that has been well-characterized. Using paramagnetic NMR techniques and X-ray absorption spectroscopy we identified an N-terminal Cu(2+) binding location and collected information on the coordination mode of the CuA site in CA, which is consistent with a four- to five-coordinate N-terminal Cu(2+) binding site reminiscent to a number of N-terminal copper(II) binding sites including the copper(II)-amino terminal Cu(2+) and Ni(2+) and copper(II)-β-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 Cu(2+) binding data to the high-affinity CuA site, we derived pH- and buffer-independent values for the thermodynamics parameters K and ΔH associated with Cu(2+) binding to the CuB site of CA to be 2 × 10(9) and -17.4 kcal/mol, respectively.