a) The aromatic dyad, F169, and F395 form a doorway that controls the water aqueduct that stretches to the heme site. Mutation of the F169A opens the doorway and allows water influx to the heme site. QM/MM calculations show that the presence of water c) The study provides a peculiar mechanistic insight that the tight binding of the substrate (S-lignin) may have an inhibitory effect on the catalytic efficiency, and the conformational flexibility is of paramount importance in successive O-demethylation reactions. Therefore, experimentally mutations in the active site binding residues such as of V241, I81and I292 in combination with the F169A can possibly alter the flexibility of the GcoA active site to accommodate various 4-alkylguaiacols hence making it even more promiscuous towards various downstream lignin by-products.