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.