Lanthanide(III)-dependent hydration of the methanol dehydrogenase cofactor, pyrroloquinoline quinone

The mechanism by which pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenases (MDH), bearing either a Ca²⁺ or a lanthanide (Ln³⁺⁾ ion in their active site, oxidize methanol has been intensely debated. In particular, the Ln³⁺-dependent activity of Ln-MDH remains poorly understood. The lack of experimental evidence represents a significant limitation to improve our understanding of these enzymes. In this work, we propose that insights on Ca- and Ln-MDH reactivity can be gained by examining a model reaction, the hydration of PQQ. Indeed, this reaction is similar to the first step of the putative methanol addition-elimination mechanism and is expected to be similarly influenced by the metal ion. The apparent affinity constants of PQQ for Ca²⁺ and Ln³⁺ were determined by UVvis absorption spectroscopy. Ln-PQQ complexes in aqueous solution were analyzed by steady-state and time-resolved fluorescence spectroscopy. The thermodynamic and kinetic parameters describing the equilibrium were obtained by variable-temperature and proton exchange spectroscopy (EXSY) NMR, as well as DFT calculations. Results demonstrated a Ln-dependent exchange rate for PQQ hydration equilibrium, the late and more Lewis acidic Ln³⁺ having the stronger impact.