Revealing the intricate mechanism governing the pH-dependent activity of a quintessential representative of flavoproteins, glucose oxidase.

Glucose oxidase (Gox), a prototypical flavoprotein, exhibits diverse industrial applications in glucose sensing and gluconic acid production. Its enzymatic activity is pH-dependent, with maximum activity observed at approximately neutral pH but less than 5% of peak activity at pH ≤ 3.0. However, the underlying mechanism governing these pH-dependent changes in activity remains elusive. Therefore, our objective was to investigate conformational alterations in Gox across different pH levels for engineering purposes. Our mutagenesis results suggest that protein degradation does not primarily contribute to the enzyme's pH-dependent activity. Fluorescence spectroscopy findings reveal subtle influences of pH on Gox's conformation while maintaining a similar overall microenvironment. Furthermore, the crystal structure and molecular dynamics simulations reveal that alterations in pH have a significant impact on the conformation of His514, a crucial catalytic residue for Gox function. These changes also result in structural variations within the substrate-binding pocket for both flavin adenine dinucleotide (cofactor) and β-d-glucose (substrate) between pH 6.0 and 2.5. Consequently, under acidic conditions (pH 2.5), β-d-glucose exhibits unstable binding within this pocket, leading to rapid dissociation from the active site. In summary, our findings underscore the intimate relationship between the conformational dynamics of His514 and the pH-dependent reaction mechanism, offering valuable insights for engineering acid-active Gox variants.