Scheme 4.

The four system configurations used for free energy calculations by alchemical transformations and thermodynamic integration via MD simulations. (A) and (C) represent the 2OG offline binding configuration in the absence (A) or the presence (C) of the substrate. (B) and (D) represent the 2OG online binding configuration in the absence (B) or the presence (D) of the substrate. For each system configuration free energy difference (ΔG_i, i = 1–4) between the H₂O bound state and the O₂ bound state was calculated.