Ab Inito Modeling of Ethylbenzene Dehydrogenase Reaction Mechanism

摘要

Abstract: Density functional theory calculations were performed to study the mechanism of ethylbenzenenoxidation by ethylbenzene dehydrogenase (EBDH). EBDH is a bacterial molybdopterin enzyme capable ofnstereospecific anaerobic hydroxylation of alkylaromatic compounds to secondary alcohols. It is a keynbiocatalyst in the metabolism of ethylbenzene-degrading bacteria such as Aromatoleum aromaticum, whichnconverts ethylbenzene to (S)-1-phenylethanol. The recently determined EBDH structure enabled thentheoretical description of the ethylbenzene oxidation mechanism. In this work, theoretical calculations andnkinetic isotopic experiments were conducted and combined in order to elucidate the reaction mechanism.nWe considered three aspects: (i) Does the reaction concur with one two-electron or two one-electronntransfers? (ii) Is the active site His192 important for the reaction and what is its protonation state? (iii)nWhat catalytic consequences have different possible arrangements of the molybdopterin ligand? The mostnimportant outcome of the calculations is that mechanisms involving two one-electron transfers and a radical-ntype intermediate have lower energy barriers than the corresponding two-electron transfer mechanismsnand are, therefore, more plausible. The mechanism involves two transition states: radical-type TS1nassociated with the C H bond cleavage, and carbocation-type TS2 associated with the transfer of thensecond electron and OH rebound. Using models with protonated and nonprotonated His 192, we concludenthat this amino acid takes part in the mechanism. However, as both models yielded plausible reactionnpathways, its protonation state cannot be easily predicted. Qualitative agreement was reached betweennthe calculated kinetic isotope effects (KIE) obtained for radical TS1 and the KIE measured experimentallynat optimum pH, but we observed a very strong pH dependence of KIE throughout the investigated pHnrange (3.1 for pH 6, 5.9 for pH 7, up to 10.5 at pH 8.). This may be explained by assuming a gradual shiftnof the rate-determining step from TS1 associated with high KIE to TS2 associated with low KIE with lowerednpH and an increasing contribution of proton/deuteron tunneling associated with high pH. Finally, modelsnwere calculated with different signs of the conformational twist of the pterin ligands, yielding only slightlyndifferent energy profiles of the reaction pathways.