Chemically, Lignin is a recalcitrant polymer that is composed of three alcoholic units: pcoumaryl alcohol (H-lignin), coniferyl alcohol (G-lignin), and synapyl alcohol (S-lignin) (Scheme 1A). The catabolism of G and S units (here represented by guaiacol and syringol, a G and S unit monomers derived from lignin) into biomass products is particularly of importance as both are major components of lignocellulose biomass. This catabolic reaction is achieved through aryl-O-demethylation (Scheme 1B) via the action of Cpd I (Fe(IV)=O porphyrin radical cation) which is also the main oxidant in P450 catalysed reactions. The reaction is initiated by the oxo group of Cpd I, by abstracting a hydrogen atom from the sp hybridised C-H bond of the methoxy group of the substrate (Scheme 2). This is followed by the rebound step, where the hydroxyl group is transferred to the substrate to form a hemiacetal product. The previous gas phase DFT calculations with implicit solvent corrections have identified the hydrogen atom abstraction (HAA) as the rate limiting step and a barrierless rebound step for both the guaiacol and syringol substrates. Unfortunately, the native P450GcoA enzyme shows the aryl-O-demethylases activity towards the guaiacol only, but not against the syringol. This creates an initial roadblock to achieving a greener and sustainable downstream lignin biodegradation through a natural enzymatic process. Very recently, using a site-directed bioengineering approach, Machovina et al engineered the native P450GcoA 19 by mutating the bulky Phenylanaline at 169 position into a smaller Alanine and achieved promising activity for the syringol (which carries an additional methoxy group in comparison to guaiacol), while it increases the native activity towards the transformation of the guaiacol as well. X-ray structure of F169A in complex with syringol show the binding of the syringol is identical to the guaiacol binding in the wild type P450GcoA. 19 20 In order to explain the experimental findings, the MD simulations on F169A and wild type P450GcoA in complex with syringol and guaiacol concluded that the Phenylalanine residues (F75, F169, and F395) helps to position the substrate in the appropriate orientation for demethylation reaction and the mutation of F169 to alanine creates an extra space that allows for the rotation of the substrate in a favorable orientation for the demethylation of syringol. However, the underlying structure feature that accounts for the promiscuous activity of P450GcoA to accommodate different lignin-derived compounds, is still not largely unclear. The previous literature on the wild type P450GcoA in complex with substrate guaiacol or product catechol induces a partially open and closed conformation of the substrate access channel. However, F169A in complex with guaiacol and syringol showed an increased probability of an open substrate access channel and exposes the active site of P450GcoA to the bulk solvent. Previous DFT calculations on the HAA and the rebound step of guaiacol or syringol in complex P450GcoA and its variant F169A neglected the effect of open substrate access channel, the role of solvent in the catalysis and the demethylation of the second methoxy group of syrnigol.