Building reactive copper centers in human carbonic anhydrase II

Abstract

Reengineering 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 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 enzymatic systems. Human carbonic anhydrase II is known to bind two Cu(2+) ions; these binding events were explored using modern isothermal titration calorimetry techniques that have become a proven method to accurately measure metal-binding thermodynamic parameters. The two Cu(2+)-binding events have different affinities (K a approximately 5 × 10(12) and 1 × 10(10)), and both are enthalpically driven processes. Reconstituting these Cu(2+) sites under a range of conditions has allowed us to assign the Cu(2+)-binding event to the three-histidine, native, metal-binding site. Our initial efforts to characterize these Cu(2+) 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.

Figure 1

Metal binding site of dicopper carbonic anhydrase. The native metal-binding site occupied with Cu²⁺ (highlighted in pink), whereas the novel, second, Cu²⁺-binding site is associated with the N-terminus (highlighted in blue). Coordinates were used from the 1.62 Å structure, 1RZC.pdb.

Figure 2

EPR spectra of 0.85 mM apoCa, 25 mM ACES, pH 7, with (A) 1.5 eq. Zn²⁺, 0.75 eq of Cu²⁺; (B) 1.9 eq of Cu²⁺. (C) Difference spectrum B-A. Microwaves: 20 μW at 9.64 GHz. Temperature: 21 K. The simulations (black lines) are least-squares fits for S=1/2, I = 3/2, with g and A_z and concentrations given in text.