Previous studies have shown the PMF values qualitatively fit with the theoretical and experimental binding affinity in case of biomolecules. The PMF, depicted in Figure , reveals that the CO2 movement trajectory from hydrophilic to hydrophobic region can be comprehended by considering four distinct states (S1  S4), as illustrated in Figure . While moving from S1  S2 state, we see a sharp increase in energy (3.8 kcal/mol), which corresponds to the disruption of CO2's hydrogen bonding with neighboring residues (His584, Ser586, Tyr503, Tyr552, and a water molecule), leading to its dissociation from the hydrophilic region S1 (cf. Figure ). Thereafter, CO2 approaches the gatekeeper residue Phe504 in the boundary region denoted as S2 (cf. Figure ). In line with the findings from the unbiased MD simulations, Phe504 plays a crucial role by changing its orientation from parallel to perpendicular (S3) (cf. Figure ), requiring an energy expenditure of approximately 3.1 kcal/mol relative to S1. This conformational change enables CO2 diffusion into the energetically more favorable hydrophobic zone that is region S4 (cf. Figure ). Upon reaching the hydrophobic zone, the gatekeeper residue Phe504 returns to its original position in preparation for the subsequent transfer of the next CO2. In summary, it is evident that the maximum energy required for the zone transition of CO2 is ~3.8 kcal/mol. Thus, from the energetic perspective, this observation suggests a highly feasible transition of CO2, which again validates our results obtained in the unbiased MD simulation.