The MD simulations of the WT P450GcoA enzyme in presence of guaiacol show two different conformations based on the substrate access channel of the enzyme (see Figure ) one is open and the other is closed conformations. In the majority of the MD trajectories (S.I In this conformation (i.e., major conformation), the substrate guaiacol was oriented in a vertical position relative to the porphyrin ring of Cpd I similar to the crystallographic structure and the previous computational studies. We found substrate binding site is mainly occupied by an aromatic triad composed of F75, F169, and F395 and several additional hydrophobic residues such as I81, V241, L244, and I292. The role of the aromatic triad has been proposed as crucial for substrate orientation. Interestingly, during simulations, none of the Phenylalanine residues of the aromatic triad (F75, F169, and F395) form direct contact with the substrate which indicates the aromatic triad does not directly participate in the catalytic activity. Furthermore, the backbone oxygen of V241 forms a strong and persistent hydrogen bond with the hydroxy group of the guaiacol which places the methoxy group in an optimal orientation for the hydroxylation via the heme center and is consistent with the previous literature. The hydrophobic triad F75, F169, and F395 were in a locked position in the substrate access channel and do not show flexibility during entire MD simulations. Interestingly, the F169 and F395 act as gates of a doorway that regulates the water inflow to the active site. In the majority of the MD trajectories, this doorway remains closed due to the locked-in position of F169 and F395 residues, blocking the access of water molecules to the substrate/heme center (Figure ).