Computational Insights into the Hydroxylation Mechanism of Toluene Catalyzed by Non-Heme Diiron Toluene 4-Monooxygenase T4moH

Abstract

Toluene monooxygenases (TMOs) are kind of diiron enzymes that catalyze the hydroxylation of aromatic rings. Four representative systems have been reported for toluene monooxygenases, including 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 the electrophilic aromatic substitution (EAS) mechanism, in which the electrophile attacks on the aromatic π-system to produce a σ-complex, followed by removal of a proton and aromatization. However, recent study on T4MO suggested an alternative reaction pathway, i.e., before the attack of the electrophile (peroxo intermediates) to the substrate, the toluene previously donates an electron to the diiron center, generating the toluene cation; then, the iron-coordinated dioxygen attacks on the para-carbon of toluene cation to carry out the hydroxylation. To clarify the T4MO-catalyzed hydroxylation mechanism of toluene and the region-selectivity, based on the crystal structure of the hydroxylase component (T4moH), we built two reactant models, and performed a series of QM/MM calculations. Our results revealed that the T4moH-catalyzed hydroxylation is triggered by μ-1,1-peroxo that contains a trivalent iron ion and a divalent iron ion, and the reaction follows the electrophilic aromatic substitution mechanism. Before the electrophilic attack, the toluene does not transfer an electron to the diiron center, as previously suggested. Instead, after the formation of tetrahedral intermediate, one electron transfers from the dirion center to the substrate moiety to promote the O-O cleavage. In addition, the μ-η 2 :η 2 -peroxo species was calculated to be a weak electrophile for attacking the aromatic ring, and the highly reactive specie of Fe IV =O is not easy to generate in the active site. The pocket Glu104 plays an important role for mediating the proton transfer in the final aromatization.These findings may provide useful information for understanding the catalysis of toluene 4-monooxygenase and carbon cycle.