Computational insights into the hydroxylation mechanism of toluene catalyzed by non-heme diiron toluene 4-monooxygenase (T4moH)

Abstract

Toluene monooxygenases (TMOs) are a type of diiron enzymes that catalyze the hydroxylation of aromatic rings. Four representative toluene monooxygenase systems have been reported, namely, ToMO from Pseudomonas stutzeri, T2MO from Burkholderia, T3MO from Ralstonia and T4MO from Pseudomonas mendocina. In the past, it was widely accepted that the TMO-catalyzed hydroxylation of arene follows an electrophilic aromatic substitution (EAS) mechanism, in which the electrophile attacks the aromatic π-system to produce a σ-complex, followed by the removal of a proton and aromatization. However, a recent study on T4MO suggested an alternative reaction pathway, i.e., before the electrophilic attack (by peroxo intermediates) on the substrate, toluene donates an electron to the diiron center, generating a toluene cation; thereafter, the iron–coordinated dioxygen attacks the para-carbon of the toluene cation to carry out the hydroxylation. To clarify the T4MO-catalyzed hydroxylation mechanism of toluene and its regioselectivity, based on the crystal structure of the hydroxylase component (T4moH), we built two reactant models and performed a series of QM/MM calculations. Results revealed that the T4moH-catalyzed hydroxylation is triggered by a μ-1,1-peroxo group that contains a trivalent iron ion and a divalent iron ion, and the reaction follows an electrophilic aromatic substitution mechanism. Before the electrophilic attack, toluene does not transfer the electron to the diiron center, as previously mentioned. Instead, after the formation of the tetrahedral intermediate, one electron is transferred from the diiron center to the substrate moiety to promote the O–O cleavage. In addition, the µ-η²:η²-peroxo species is a weak electrophile for attacking the aromatic ring, and the highly reactive Fe^IV = O species is not easy to generate in the active site. The pocket Glu104 plays an important role in mediating proton transfer in the final aromatization. These findings may provide useful information for understanding the catalysis of toluene 4-monooxygenase and the carbon cycle.