Catalysis of Electron Transfer during Activation of O2 by the Flavoprotein Glucose Oxidase

Two prototropic forms of glucose oxidase undergo aerobic oxidation reactions that convert FADH- to FAD and form H2O2 as a product. Limiting rate constants of kcat/KM(O2) = (5.7 ± 1.8) × 102 M-1· s-1 and kcat/KM(O2) = (1.5 ± 0.3) × 106 M-1· s-1 are observed at high and low pH, respectively. Reactions exhibit oxygen-18 kinetic isotope effects but no solvent kinetic isotope effects, consistent with mechanisms of rate-limiting electron transfer from flavin to O2. Site-directed mutagenesis studies reveal that the pH dependence of the rates is caused by protonation of a highly conserved histidine in the active site. Temperature studies (283-323 K) indicate that protonation of His-516 results in a reduction of the activation energy barrier by 6.0 kcal· mol-1 (0.26 eV). Within the context of Marcus theory, catalysis of electron transfer is attributed to a 19-kcal· mol-1 (0.82 eV) decrease in the reorganization energy and a much smaller 2.2-kcal· mol-1 (0.095 eV) enhancement of the reaction driving force. An explanation is advanced that is based on changes in outer-sphere reorganization as a function of pH. The active site is optimized at low pH, but not at high pH or in the H516A mutant where rates resemble the uncatalyzed reaction in solution.