Computational Insights into the Non-Heme Diiron Alkane Monooxygenase Enzyme AlkB: Electronic Structures, Dioxygen Activation, and Hydroxylation Mechanism of Liquid Alkanes

Alkane monooxygenase (AlkB) is a membrane-spanning metalloenzyme that catalyzes the terminal hydroxylation of straight-chain alkanes involved in the microbially mediated degradation of liquid alkanes. According to the cryoEM structures, AlkB features a unique multihistidine ligand coordination environment with a long Fe-Fe distance in its active center. Up to now, how AlkB employs the diiron center to activate dioxygen and which species is responsible for triggering the hydroxylation are still elusive. In this work, we constructed computational models and performed quantum mechanics/molecular mechanics (QM/MM) calculations to illuminate the electronic characteristics of the diiron active center and how AlkB carries out the terminal hydroxylation. Our calculations revealed that the spin-spin interaction between two irons is rather weak. The dioxygen may ligate to either the Fe1 or Fe2 atom and prefers to act as a linker to increase the spin-spin interaction of two irons, facilitating the dioxygen cleavage to generate the highly reactive Fe(IV)═O. Thus, AlkB employs Fe(IV)═O to trigger the hydrogen abstraction. In addition, the previously suggested mechanism that AlkB uses both the dioxygen and Fe-coordinated water to perform hydroxylation was calculated to be unlikely. Besides, our results indicate that AlkB cannot use the Fe-coordinated dioxygen to directly trigger hydrogen abstraction.