Kinetic parameters of the oxidative part of glucose-oxidase reaction have been measured with 16 different electron-acceptors and glucose as a substrate. 2. In each case, the rate-limiting portion of the oxidative part of reaction was the formation of the E-FADH2.Acceptor-complex; this rate was pH-independent around the pH-optimum of the enzyme. 3. In each case, E-FADH2 acceptor-complex was undetectable in the steady-state kinetics, with the exception of cytochrome-c. 4. The rates of redox reactions between various forms of reduced 5-ethyl-lumiflavin and five different electron-acceptors have been examined with a conventional spectrophotometry. In each case, it was found that the reactions proceeded at high rates whenever thermodynamically feasible, and were totally prevented in the opposite case. 5. Molecular oxygen was able to oxidize only the neutral form of 5-ethyl-1,5-dihydrolumiflavin to its radical form, at a moderate rate; all other forms of reduced 5-ethyl-lumiflavin were not oxidized by O2. 6. By the comparison of enzymatic and model redox reactions, it was possible to establish the minimal mechanism of the oxidative part of the glucose-oxidase catalytic cycle.
