An RDF plot showing the interaction between water and Tyr476 is also provided in the SI (cf. Figure ). In addition, we have plotted the water population at various radii from the hydroxyl group of the Tyr476 residue, illustrating the consistent nature of the water chain near this hydroxyl group (cf. Figure ). Given the challenges of computationally estimating the absolute pKa of a residue, we instead estimated the pKa shift of Tyr476 due to hydrogen bonding interactions with nearby water molecules, using a protocol recently employed by the Wang group . As shown in Figure , our calculations reveal that the pKa of Tyr476 can be lowered by ~2.86 units from its absolute value of 10.1, resulting in a pKa of 7.24 in the enzymatic environment. This lowered pKa favors the deprotonated form of Tyr476 at the experimental pH, making it a viable proton acceptor residue during catalysis of CO2 hydration. , the Tyr476 residue is wellconnected to CO2 through three water molecules (W1, W2, and W3), with intermolecular distances ranging from 1.6 to 1.8 Å, indicating strong hydrogen bonding interactions. We note that such an organized water chain was absent in the crystal structure and is observed only during the catalytic process. Since metal-free CA lacks a catalytic group analogous to those in metal-containing CA that can stabilize OH -after proton abstraction (cf. Scheme 1), the reaction must proceed via a concerted pathway. We therefore performed a PES scan starting from the RC (cf. Figure ). The reaction initiates with the deprotonated Tyr476 abstracting a proton from W3, generating the reactive OH -. This OH -is then stabilized by the nearby W2. This process continues similarly to the 'Grotthuss proton hopping' mechanism, eventually generating the OH -from W1, which then nucleophilically attacks the carbon atom of CO2 to produce HCO3 -. All these processes occur simultaneously, as illustrated in Figure . In addition, a video of this complete transformation is provided in the SI (cf. Video VS2). The energies are presented relative to the RC in kcal/mol, calculated using the B3LYP-D3/def2-TZVP level of theory. All energies are ZPE corrected. We note that RC, TS, and PC represent 'reactant complex,' 'transition state,' and 'product complex,' respectively.