The study by Machvina et al shows that the F169A mutant significantly affects the syringol activity, changing CYP450GcoA from a specialized enzyme to a promiscuous one. We, therefore, simulated the F169A mutant in a complex with syringol. Interestingly, two conformations of the substrate (Figure ) were observed which are in contrast to the previous literature and indicates the importance of sampling in studying enzyme catalysis. In both conformations, one methoxy group was positioned proximal to the Fe (IV)=O while the other stays away with a large distance. Unlike WT complex with syringol, no C-H-π nor C-H-O interaction of the substrate with protein residues was observed due to the lack of benzene ring in the mutant and the large distance between the substrate and the residue at position 169. Although the key active site residues maintain the same orientations in conformation A and B, the orientation of the substrate significantly differs in both conformations (see arrows in Figure ). Due to the different orientations of syringol, it binds tightly with Val241 in conformation A while it stays away in conformation B. Furthermore, a persistent water channel was found in conformation B that stretches between A169 and F395 residues and it solvates the substrate and the active site. Due to the lack of a stabilizing interaction in conformation B, the substrate shows a high degree of conformational mobility that may facilitate the second oxidation of the intermediate 3MC.