Transient Formation of a Neutral Ubisemiquinone Radical and Subsequent Intramolecular Electron Transfer to Pyrroloquinoline Quinone in theEscherichia coliMembrane-Integrated Glucose Dehydrogenase

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Abstract
The membrane-bound quinoprotein glucose dehydrogenase (mGDH) in Escherichia coli contains pyrroloquinoline quinone (PQQ) and participates in the direct oxidation of d-glucose to d-gluconate by transferring electrons to ubiquinone (UQ). To elucidate the mechanism of ubiquinone reduction by mGDH, we applied a pulse radiolysis technique to mGDH with or without bound UQ8. With the UQ8-bound enzyme, a hydrated electron reacted with mGDH to form a transient species with an absorption maximum at 420 nm, characteristic of formation of a neutral ubisemiquinone radical. Subsequently, the decay of the absorbance at 420 nm was accompanied by an increase in the absorbance at 370 nm. Experiments with the PQQ-free apoenzyme showed no such subsequent absorption changes, although ubisemiquinone was formed. These results indicate that a pathway for an intramolecular electron transfer from ubisemiquinone radical at the UQ8 binding site to PQQ exists in mGDH. The first-order rate constant of this process was calculated to be equal to 1.2 × 103 s-1. These findings are consistent with our proposal that during the catalytic cycle of mGDH the bound UQ8 mediates electron transfer from the reduced PQQ to UQ8 pools.

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