Carbonic anhydrase (CA) is a well-studied, zinc-dependent metalloenzyme that catalyzes the hydrolysis of carbon dioxide to the bicarbonate ion. The apo-form of CA (apoCA) is relatively easy to generate, and the reconstitution of the human erythrocyte CA has been initially investigated. In the past, these studies have continually relied on equilibrium dialysis measurements to ascertain an extremely strong association constant (Ka ~ 1.2×1012) for Zn2+. However, new reactivity data and isothermal titration calorimetry (ITC) data reported herein call that number into question. As shown in the ITC experiments, the catalytic site binds a stoichiometric quantity of Zn2+ with a strong equilibrium constant (Ka ~ 2 × 109) that is three orders of magnitude lower than the previously established value. The thermodynamic parameters associated with Zn2+ binding to apoCA are unraveled from a series of complex equilibria associated with the in vitro metal binding event. This in-depth analysis adds clarity to the complex ion chemistry associated with zinc binding to carbonic anhydrase and validates thermochemical methods that accurately measure association constants and thermodynamic parameters for complex-ion and coordination chemistry observed in vitro. Additionally, the zinc sites in both the as-isolated and reconstituted ZnCA were probed using X-ray absorption spectroscopy. Both X-ray absorption near edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) analyses indicate the zinc center in the reconstituted carbonic anhydrase is nearly identical to that of the as-isolated protein and confirms the notion that the metal binding data reported herein is the reconstitution of the zinc active site of human CA II.

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 two equivalents of copper(II) with high affinity. The Cu2+ ions bind independently forming two non-coupled 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 have identified an N-terminal Cu2+ 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 Cu2+ binding site reminiscent to a number of N-terminal copper(II) binding sites including the copper(II)-ATCUN 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 CuA site, we have derived pH- and buffer-independent values for the thermodynamics parameters K and ΔH associated with Cu2+ binding to the CuB site of CA to be 2 × 109 and −17.4 kcal/mol, respectively.

Re-engineering metalloproteins to generate new biologically relevant metal centers is an effective a way to test our understanding of the structural and mechanistic features that steer chemical transformations in biological systems. Here we report thermodynamic data characterizing the formation of two type-2 (T2) copper sites in carbonic anhydrase and experimental evidence showing one of these new copper centers has characteristics similar to a variety of well-characterized copper centers in synthetic models and in enzymatic systems. Human CA II is known to bind two Cu2+ ions; herein, these binding events are explored using modern isothermal titration calorimetry (ITC) techniques that have become a proven method to accurately measure metal-binding thermodynamic parameters. The two Cu2+-binding events have different affinities (Ka ∼ 5 × 1012 and 1 × 1010) and both are enthalpically driven processes. Reconstituting these Cu2+ sites under a range of conditions has allowed us to assign the Cu2+-binding event to the three-histidine, native, metal binding site. Our initial efforts to characterize these Cu2+ sites have yielded data that show distinctive (and noncoupled) EPR signals associated with each copper-binding site, and that this reconstituted enzyme can activate hydrogen peroxide to catalyze the oxidation of 2-aminophenol.

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