The findings of this study elucidate the identification of the catalytic site and the mechanism of CO2 hydration in metal-free CA. Our calculations reveal that, despite HCO3 -being cocrystallized in the hydrophilic zone of the enzyme, the catalytic site is located in the hydrophobic zone. The strategic Phe504 residue regulates CO2 transportation from the hydrophilic to the hydrophobic zone in an energetically favorable manner, serving as a gatekeeper that allows only CO2 to transfer and not HCO3 -. Once CO2 reaches the hydrophobic zone, it converts to HCO3 -in situ; however, HCO3 -exits the site rapidly, eliminating the possibility of dehydration. Our hybrid QM/MM calculations demonstrate that CO2 hydration is energetically feasible in the hydrophobic zone with a deprotonated Tyr residue acting as a proton acceptor. All the mechanistic study holds true for both Bn86287 and all2909 enzymes.