Mechanism of methanol oxidation by quinoprotein methanol dehydrogenase

At neutral pH, oxidation of CH₃OH → CH₂O by an o-quinone requires general-base catalysis and the reaction is endothermic. The active-site -CO₂⁻ groups of Glu-171 and Asp-297 (Glu-171-CO₂⁻ and Asp-297-CO₂⁻) 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-CO₂⁻ and the crystal water (Wat1) is hydrogen bonded to Asp-297-CO₂⁻ is ΔG^‡ = 11.7 kcal/mol, which is comparable with the experimental value of 8.5 kcal/mol. The calculated ΔG^‡ when MeOH is hydrogen bonded to Asp-297-CO₂⁻ is >50 kcal/mol. The Asp-297-CO₂⁻...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-CO₂⁻ bound Wat1 for a solvent water. Starting with Wat1 removed and MeOH hydrogen bonded to Asp-297-CO₂⁻, we find that MeOH returns to be hydrogen bonded to Glu-171-CO₂⁻ and Asp-297-CO₂⁻ coordinates to Ca²⁺ during 3 ns simulation. The Asp-297-CO₂⁻...Wat1 of reactant complex does play a crucial role in catalysis. By QM/MM calculation ΔG^‡ = 1.1 kcal/mol for Asp-297-CO₂⁻ general-base catalysis of Wat1 hydration of the immediate CH₂O product → CH₂(OH)₂. By this means, the endothermic oxidation-reduction reaction is pulled such that the overall conversion of MeOH to CH₂(OH)₂ is exothermic.