Determination of a large reorganization energy barrier for hydride abstraction by glucose oxidase.

Glucose oxidase mediates the aerobic oxidation of simple sugars to lactones using a noncovalently bound flavin cofactor. The chemical mechanism of this reaction has been uncertain for many years. Here it is shown, using enzymes reconstituted with chemically modified cofactors, that sugar oxidation most likely occurs by concerted hydride (H-) abstraction. Studies of the kinetics and thermodynamics together with the application of Marcus theory reveal a large reorganization energy barrier. The magnitude of this intrinsic contribution appears characteristic of H- transfer in proteins and in solution. The observation that neither the thermodynamics nor reorganization energy is significantly altered in the glucose oxidase active site raises questions concerning how the redox reaction may be catalyzed.