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Abstract

There is a long-standing mechanistic consensus that alcohol oxidation by cytochrome P450 enzymes is triggered by hydrogen abstraction from the α-C–H bond of the alcohol. Through combined molecular dynamics simulations and quantum mechanics/molecular mechanics calculations we demonstrate that this is not the case in P450 2E1-mediated ethanol oxidation. We show that while the O–H bond is stronger than the α-C–H bonds in alcohols, the intrinsic reactivity of O–H and α-C–H bonds is comparable for hydrogen abstraction, due to the strong electrostatic interaction between the ethanol hydroxyl group and the Fe═O moiety. Thus, the binding of ethanol to the Fe═O moiety in the P450 2E1 pocket is of particular importance to the reaction mechanism. We further show that the Thr303 residue plays a crucial role in confining the ethanol substrate in the active site of P450 2E1 and thereby steering the initial hydrogen abstraction from the O–H bond of ethanol. Because of the highly endothermic O–H bond cleavage, the subsequent hydrogen abstraction of α-C–H bond is the overall rate-determining step for ethanol oxidation. These mechanistic findings are in agreement with available experimental data (e.g., kinetic isotope experiments and electron spin resonance analysis). Our work sheds light on the puzzling mechanism of ethanol oxidation in P450 2E1 by identifying the directing effect of Thr303 on substrate orientation, which complements its role as a proton-shuttle mediator during the formation of Compound I.