To evaluate the effect of the aromatic triad consisting of F75, F169, and F395, which was suggested to help to position the substrate in a catalytically competent orientation, we performed another set of QM/MM calculations using a different MD snapshot of WT P450GcoA with the extended QM region to include the aromatic triad. The reaction barrier for HAA from Cpd I for this snapshot displayed a similar reaction barrier of 19.5 kcal/mol (Figure and Table ). The results are consistent with the QM/MM calculations with a smaller QM region, which indicates the aromatic triad does not directly participate in the catalytic activity but determines the substrate recognition and binding. The benchmarking was also done with the def2-TZVP basis set, which yielded a similar barrier to that obtained by using def2-SVP.