Mechanism of methanol oxidation by quinoprotein methanol dehydrogenase

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Abstract
At neutral pH, oxidation of CH 3 OH → CH 2 O by an o -quinone requires general-base catalysis and the reaction is endothermic. The active-site –CO 2 − groups of Glu-171 and Asp-297 (Glu-171–CO 2 − and Asp-297–CO 2 − ) have been considered as the required general base catalysts in the bacterial o -quinoprotein methanol dehydrogenase (MDH) reaction. Based on quantum mechanics/molecular mechanics (QM/MM) calculations, the free energy for MeOH reduction of o -PQQ when MeOH is hydrogen bonded to Glu-171–CO 2 − and the crystal water (Wat1) is hydrogen bonded to Asp-297–CO 2 − is Δ G ‡ = 11.7 kcal/mol, which is comparable with the experimental value of 8.5 kcal/mol. The calculated Δ G ‡ when MeOH is hydrogen bonded to Asp-297–CO 2 − is >50 kcal/mol. The Asp-297–CO 2 − ···Wat1 complex is very stable. Molecular dynamics (MD) simulations on MDH·PQQ·Wat1 complex in TIP3P water for 5 ns does not result in interchange of Asp-297–CO 2 − bound Wat1 for a solvent water. Starting with Wat1 removed and MeOH hydrogen bonded to Asp-297–CO 2 − , we find that MeOH returns to be hydrogen bonded to Glu-171–CO 2 − and Asp-297–CO 2 − coordinates to Ca 2+ during 3 ns simulation. The Asp-297–CO 2 − ···Wat1 of reactant complex does play a crucial role in catalysis. By QM/MM calculation Δ G ‡ = 1.1 kcal/mol for Asp-297–CO 2 − general-base catalysis of Wat1 hydration of the immediate CH 2 O product → CH 2 (OH) 2 . By this means, the endothermic oxidation-reduction reaction is pulled such that the overall conversion of MeOH to CH 2 (OH) 2 is exothermic.

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