A previous study by Machvina et al shows F169A slightly enhanced activity toward the guaiacol , and our simulation shows that F169A mutation causes increased water inflow to the heme center. Hence, whether water plays a role in the catalysis, must be elucidated. Therefore, we performed QM/MM calculations for the WT and F169A mutant in order to understand the enhanced activity. We started our QM/MM calculations with representative snapshots of WT and F169A mutant complexes taken from the most populated snapshots from the MD simulations and performed the PES scanning of the QM/MM optimized structures. The reaction profiles and the reactive species for the O-demethylation at methoxy carbon for WT and F169A mutant (Figure ) show that the oxidation of the guaiacol is initiated by a hydrogen atom abstraction (HAT) via the Cpd I and goes through a transition state TS. This is in accordance with the previous DFT calculations. Interestingly, the methyl hydrogen of the guaiacol in the WT complex is positioned at 2.12 Å and is closer to the oxo moiety of Cpd I than the methyl hydrogen of the mutant at 2.21 Å. However, the HAA barrier in the mutant (15.8 kcal/mol) is lower than the HAA barrier in the WT complex (19.4 kcal/mol). We found the additional water in the F169A mutant plays a pivotal role in stabilizing the transition state (at 1.18 Å) relative to the transition state (at 1.16 Å) in the WT complex. The role of water near the Cpd I in stabilising the transition state associate with the HAA through electrostatic interactions is well documented by Shaik and coworkers. The IM1 in both complexes are endothermic by a similar energy value and possess a similar geometry. Subsequently, the hydroxyl group is transferred to the substrate via a rebound mechanism and the hydroxylated product is formed in a barrierless process in both WT and mutant complexes. These results are in good agreement with the experimental data that the F169A mutant increases the guaiacol activity. The QM/MM reaction profile was computed using U-B3LYP functional with D3BJ dispersion correction and the def2-SVP basis set for S=1/2. The data denotes the relative energies with added ZPE (kcal/mol). The structures on the S=1/2 are shown in a 2D sketch. (B) The stationary point and transition structures associated with the oxidation of guaiacol in the active site of WT (upper panel, porphyrin ring shown in green) and F169A P450GcoA enzyme (lower panel, porphyrin ring shown in pink). A water that is in the proximity of iron oxo in the F169A variant is shown by stick mode. The key distances are shown in (Å), the angle of hydrogen atom abstraction is shown in degree.