Catalysis of electron transfer during activation of O2 by the flavoprotein glucose oxidase

Abstract

Two prototropic forms of glucose oxidase undergo aerobic oxidation reactions that convert FADH(-) to FAD and form H(2)O(2) as a product. Limiting rate constants of k(cat)K(M)(O(2)) = (5.7 +/- 1.8) x 10(2) M(-1).s(-1) and k(cat)K(M)(O(2)) = (1.5 +/- 0.3) x 10(6) 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 O(2). 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.

Fig 1.

(Left) Profiles of log k_cat/K_M(O₂) for WT (○) and the H516A (□) versus pH at 298 K and μ = 0.1 M. Rate constants fitted to the expression log k_cat/K_M(O₂) = log[k_Min/(1 + 10^{pKa − pH}) + k_Max/(1 ± 10^{pH − pKa})] are given in Table 1. (Right) Temperature dependencies of ln k_cat/K_M(O₂) for WT at pH 5.0 (○) and pH 12.5 (□). Activation parameters are given in Table 1.

Fig 2.

Active-site residues and conserved water molecule relative to the bound flavin in oxidized GO. (Structure from ref. .)