Here we investigated the binding and catalytic mechanism of the promiscuity for both syringol and native guaiacol, using comprehensive MD simulations to improve sampling (16 replicas * 500ns (see SI Table ) and hybrid state of the art QM/MM calculations which take into account the whole protein environment which is essential for enzyme catalysis to address the following mechanistic puzzles: a) In contrast to guaiacol doesn't change the orientation in different P450GcoA variants , why does the enzyme show prominent alternation in the active site while it is bound with syringol (S-unit)? b) whether the water in the F169A mutant plays a role in the spontaneous promiscuity for different lignin-derived compounds? c) In contrast to guaiacol, the O-demethylation of syringol occurs in two successive reaction steps and how is second methoxy group of syringol (3-Methoxycatechol) demethylated to the final product pyrogallol; Would the second reaction sphere plays a role in the promiscuous activity of the F169A mutant?