Elucidating the role of metal ions in carbonic anhydrase catalysis

Abstract

Why metalloenzymes often show dramatic changes in their catalytic activity when subjected to chemically similar but non-native metal substitutions is a long-standing puzzle. Here, we report on the catalytic roles of metal ions in a model metalloenzyme system, human carbonic anhydrase II (CA II). Through a comparative study on the intermediate states of the zinc-bound native CA II and non-native metal-substituted CA IIs, we demonstrate that the characteristic metal ion coordination geometries (tetrahedral for Zn²⁺, tetrahedral to octahedral conversion for Co²⁺, octahedral for Ni²⁺, and trigonal bipyramidal for Cu²⁺) directly modulate the catalytic efficacy. In addition, we reveal that the metal ions have a long-range (~10 Å) electrostatic effect on restructuring water network in the active site. Our study provides evidence that the metal ions in metalloenzymes have a crucial impact on the catalytic mechanism beyond their primary chemical properties.

Fig. 1. Structure of native carbonic anhydrase II (Zn-CA II) and its catalytic mechanism.

a The active site consists of zinc binding site, hydrophobic/hydrophilic regions, and entrance conduit (EC). b The water networks in the active site are responsible for the proton transfer (red) and substrate/product/water exchange (blue) during enzyme catalysis. c The CO₂ hydration reaction mechanism of Zn-CA II. First, CO₂ binds to the active site, leading to a nucleophilic attack by the zinc-bound hydroxyl ion onto CO₂. HCO₃⁻ thus formed is subsequently displaced by the water molecule inflowing through EC. The HCO₃⁻ molecule likely binds to Zn²⁺ ion in a monodentate mode and its OH group is held at the Zn²⁺ ion due to the hydrogen bonding with Thr199^, . This product binding configuration leads to a weak interaction between the product and Zn²⁺ ion, thereby facilitating fast product dissociation. Finally, proton transfer occurs via the network (W_Zn → W1 → W2 → His64) provided by the protein scaffold.

Fig. 2. Metal coordination geometry in CA II without CO₂ pressurization.

a In apo-CA II, the metal binding site is vacant. b, c Zn- and Co-CA II show tetrahedral, d Ni-CA II octahedral, and e Cu-CA II trigonal bipyramidal coordination geometry. The electron density (2F_o–F_c, blue) is contoured at 2.2σ. All structures were obtained at pH 7.8 except for (c) which is obtained at pH 11.0. The intermediate water (W_I) in (d) is colored in steel blue for clarity.

Fig. 3. Substrate/product binding in apo- and Zn-CA II.

The intermediate water (W_I) is colored in steel blue for clarity. The electron density (2F_o–F_c, blue) is contoured at 2.2σ. a, b At 20 atm of CO₂ pressure, apo-CA II shows clear binding of CO₂ without the need of Zn²⁺ ion. c Zn-CA II shows similar binding of CO₂ as in apo-CA II while maintaining tetrahedral metal coordination. d Upon CO₂ binding (white) in Zn-CA II, W_Zn is located at the center of the hypothetical tetrahedral arrangement made up of Zn²⁺ ion, Thr199-Oγ1, position (1) (close to W1), and position (2) (close to the carbon atom in CO₂). In this configuration, a hybridized lone pair in W_Zn directly faces CO₂ molecule at a distance, appropriate for efficient nucleophilic attack. Distance between the position (2) and C atom of CO₂ is merely 0.36 Å.

Fig. 4. Substrate/product binding in Co-CA II.

The intermediate water (W_I) is colored in steel blue for clarity. The electron density (2F_o–F_c, blue) and the difference map (F_o–F_c, green) are contoured at 2.2σ and 7.0σ, respectively. a, b At 20 atm of CO₂ pressure, Co-CA II at pH 11.0 shows superposition of CO₂ binding (~50% occupancy, white) with tetrahedral coordination and HCO₃⁻ binding (~50% occupancy) with octahedral coordination. c, d Co-CA II at pH 7.8 shows complete binding of HCO₃⁻, showing octahedral coordination even in absence of added CO₂. It is likely that the captured HCO₃⁻ is converted from the CO₂ absorbed in the crystal from ambient air.

Fig. 5. Substrate/product binding in Ni- and Cu-CA II.

The intermediate water (W_I) is colored in steel blue for clarity. The electron density (2F_o–F_c, blue) is contoured at 2.2σ. a At 20 atm of CO₂ pressure, Ni-CA II maintains octahedral coordination with HCO₃⁻ binding. b Compared to the W_Zn geometry in Zn-CA II (Fig. 3d), the nucleophilic attack geometry around W_Ni′ has steric hindrance on CO₂ molecule (adapted from Zn-CA II, 20 atm, white) and is distorted away. Distance between the position (2) and C atom of CO₂ is 1.55 Å. c Cu-CA II shows only disordered electron density in the CO₂/HCO₃⁻ binding site. d The nucleophilic attack geometry around W_Cu has steric hindrance on CO₂ molecule (adapted from Zn-CA II, 20 atm) and is significantly distorted away. Distance between the position (2) and C atom of CO₂ is 2.93 Å.

Fig. 6. Active site in CA II showing proton transfer pathway and EC water network (W_EC1 ~ W_EC5).

The electron density (2F_o–F_c) is contoured at 1.7σ except for EC waters at 1.5σ. The EC waters are colored in aqua marine and the intermediate waters (W_I and W_I′) in steel blue for clarity. W2′ is an alternative position of W2. The possible proton transfer pathways in the metal-CA IIs are depicted as red arrows. All structures were obtained at pH 7.8 except for e at pH 11.0. a, b Apo-CA II shows well-ordered water arrangement (dotted red line) with His64 favored in outward conformation at 0 atm CO₂ pressure. Upon CO₂ binding, His64 moves inward and water molecules show highly dynamical motions, stabilizing W_I. c, d Zn-CA II shows His64 favored in inward conformation at 0 atm CO₂ pressure. Upon CO₂ binding, W2, W3b, and W_EC waters show significantly different dynamics with His64 moving outward, and an additional intermediate water (W_I′) is stabilized with the W_EC molecules. The motions of W3b and W_EC1 turn on the dynamic interplay between the proton transfer and EC water networks. e, f Co-CA II shows similar arrangement initially as in Zn-CA II. However, upon full HCO₃⁻ binding, the dynamical motions of EC waters are different and the intermediate water W_I′ is less stabilized. Note that, in Co-CA II, proton transfer seems to occur while the product is still bound. g, h Ni-CA II initially shows altered water arrangements due to octahedral coordination. Upon HCO₃⁻ binding, significantly reduced water dynamical motions are recognized. i, j Cu-CA II shows unexpectedly similar dynamical motions of active site waters and His64 as in Zn-CA II.

Fig. 7. Proposed catalytic mechanism of Co-CA II.

In Co-CA II, CO₂ binding and the catalytic conversion of CO₂ to HCO₃⁻ occur in the same way as in the Zn-CA II with tetrahedral geometry. However, the HCO₃⁻ displacement and proton transfer process are significantly altered due to the coordination expansion to octahedral geometry during catalysis. This octahedral coordination allows bidentate binding mode of HCO₃⁻ and reorganization of negative charge of HCO₃⁻ toward Co²⁺ ion, allowing stronger HCO₃⁻ binding to metal ion. To dissociate the product, proton transfer first occurs via an altered pathway (possibly, W_Co,octa → W2 → His64) and W_Co,octa is converted into the hydroxyl ion. This negatively charged hydroxyl ion then pushes away the bound product, and the tetrahedral coordination is restored for the next catalytic cycle.

Fig. 8. Proposed catalytic mechanism of Ni-CA II.

In Ni-CA II, octahedral coordination is maintained throughout the whole catalytic cycle. The significant consequence is that one of the three bound water molecules experiences steric hindrance with the CO₂ binding. In addition, the nucleophilic attack geometry is distorted (Fig. 5b), suggesting less efficient conversion into HCO₃⁻. The formed HCO₃⁻ is strongly bound to Ni²⁺ ion in a bidentate mode as in the Co-CA II but is directly displaced by two inflowing water molecules. Finally, proton transfer occurs via an altered network (possibly, W_Ni′ → W_Ni → W2 → His64) to restore the catalytic cycle.

Fig. 9. Proposed catalytic mechanism of Cu-CA II.

In Cu-CA II, one of the two bound water molecules in the trigonal bipyramid coordination experiences significant steric hindrance from CO₂ molecule, hindering adoption of proper configuration for nucleophilic attack. In addition, even if CO₂ binds to the active site temporarily, the nucleophilic attack geometry is too distant (3.9 Å) and significantly distorted (Fig. 5d).