Characterization of the Copper(II) Binding Sites in Human Carbonic Anhydrase II

Abstract

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.

Figure 1

Copper(II) substituted carbonic anhydrase. (A) Location of the crystallographically characterized Cu²⁺ binding sites in Cu₂CA from 1RZC.pdb. Traditional metal binding site in CA is highlighted in pink, where the novel Cu-binding site is shown in yellow. (B) The novel Cu binding site in CA, proposed to be the Cu_A center. His4 and His64 in the Cu_A site are observed to have two different conformers (shown in cyan and colored ball and stick) within 1RZC.pdb.

Figure 2

¹H Γ₂ PRE rates in Zn/CuCA. A comparison of observed PRE rates for backbone protons (black points) to the predicted rates (red lines) using the optimal Cu²⁺ coordinates in Zn/CuCA. The coordinates of the optimal Cu center are shown in Fig 3. PREs were measured using the pulse program described by Anthis, et al. and coordinate optimization was performed using the previously-determined structure of HCA (PDB ID 2CBA).

Figure 3

Best-fit coordinates for Cu²⁺ bound to the Cu_A site in CA. The optimized position of the paramagnetic copper(II) ion (Cu_A site) in both Zn/CuCA and Cu₂CA is shown as a yellow spheroid. This region lacks secondary structure, and the protein was treated as a rigid body during optimization, and the size and shape of this binding site is representative of the statistical uncertainty in the data. Note the copper atom (orange) described by the previously reported crystal structure of the two copper(II) coordination (1RZC.pdb).

Figure 4

XANES spectra (A) unfiltered k³χ(k) EXAFS spectra (B), and the Fourier transform (C, k = 2.0 − 12.0 Å⁻¹) for Cu/ZnCA ensuring Cu²⁺ coordination to the Cu_A site. Experimental data are represented by dotted lines, while best fits (given in bold in Table S5) are shown as solid black lines.

Figure 5

ITC data of copper binding to apoCA. (A) Raw data from the titration of a 1.5 mL cell containing 70 µM apoCA was titrated with 80 × 3 µL of 1.48 mM Cu(NO₃)₂ in 100 mM TES at pH 7.4. (B) integrated isotherm and the best associated fit for a two-site binding model. The average thermodynamic parameters associated with Cu²⁺ binding to apoCA are reported in Table 1.

Figure 6

Plot of the addition of observed enthalpy from ITC and metal-buffer interaction enthalpy (ΔH_ITC + ∆H_Cu-Buffer) versus buffer ionization enthalpies (∆H_H-Buffer) in various buffers at pH 7.4. Values for ΔH_ITC, ∆H_Cu-Buffer and ∆H_H-Buffer in various buffers are listed in Table 3.3 and Table 3.4. Symbols shown as diamonds (Cu_A) and squares (Cu_B), and the linear regression values are y = 1.76 × − 0.68, R² = 0.99, and y = 0.82 × − 6.46, R² = 0.99, respectively.

Figure 7

A comparison of the four possible structures of the Cu_A site. The ATCUN having an N-terminal binding, two backbone amides, and histidine coordinated with the Cu ion. The ATCUN-like have an N-terminal binding, one backbone amide, a histidine, and another N/O donor ligand. The expected structure of the Cu_A site. The A-β structure with an N-terminal binding, carboxyl oxygen coordination, and two histidines. The final possibility is the ATCUN/A-β with an N-terminal binding, backbone amide, carboxyl oxygen, and histidine coordination to the copper ion.