Mechanism of methanol oxidation by quinoprotein methanol dehydrogenase

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 DeltaG++ = 11.7 kcal/mol, which is comparable with the experimental value of 8.5 kcal/mol. The calculated DeltaG++ 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 DeltaG++ = 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.

Scheme. 1.

Scheme. 2.

Fig. 1.

MD simulations. (A) Time-dependent variation of the distances between hydroxyl oxygen of MeOH and the carboxylate oxygen of Glu-171–CO₂⁻ and Asp-297–CO₂⁻, respectively. (B) The coordination of Ca²⁺ with O7A (PQQ) and the carboxylate oxygen (OD1) of Asp-297–CO₂⁻, respectively, at the ground state of MDH·PQQ·MeOH when Wat1 is replaced by MeOH.

Fig. 2.

Contour plot of the potential energy profiles using SCC-DFTB/MM and two-dimensional reaction coordinates y = r_{CB(MeOH)-HB1(MeOH)} − r_{HB1(MeOH)-C5(PQQ)} and x = r_{OG(MeOH)-HG1(MeOH)} − r_{HG1(MeOH)-OD1(Asp-297)} (A), and y = r_{CB(MeOH)-HB1(MeOH)} − r_{HB1(MeOH)-C5(PQQ)} and x = r_{OG(MeOH)-HG1(MeOH)} − r_{HG1(MeOH)-OE1(Glu-171)} (B) involving Asp-297–CO₂⁻ general base in MDH·PQQ·MeOH complex and Glu-171–CO₂⁻ general base catalyst in MDH·PQQ·MeOH·Wat1 complex, respectively. The position of the TS is marked by an asterisk.

Fig. 3.

The structure of the active site of MDH·PQQ·MeOH·Wat1 complex at the ground state (A) and the close-up of the reaction region (B) as determined by ABNR energy-minimizing the final structure of our previous 3-ns MD simulations at the SCC-DFTB/MM level.

Fig. 4.

The potential surface at the SCC-DFTB/M level (in kcal/mol) for the oxidation reaction of MeOH catalyzed by MDH (solid line) and the hydration of OCH₂ (dotted line).

Fig. 5.

The structure of the transition state (TS) (Scheme 2) at the active site (A) and the close-up of the reaction region (B) as determined by adiabatic mapping at the SCC-DFTB/MM level.

Fig. 6.

The structure of the intermediate MDH·PQQ(C5H)⁻·OCH₂·Wat1 at the active site as determined by the SCC-DFTB/MM ABNR energy-minimization method.

Scheme. 3.