| Abstract: | The reduction of D-amino acid oxidase (DAAO) by hydrated electrons (eaq-) has been studied in the absence and presence of benzoate by pulse radiolysis. The eaq-did not reduce the flavin moiety in DAAO and reacted with the amino acid residues in the protein. In the presence of benzoate, eaq- first reacted with benzoate to yield benzoate anion radical. Subsequently, the benzoate anion radical transferred an electron to the complex of DAAO-benzoate to form the red semiquinone of the enzyme with a second-order rate constant of 1.2 X 10(9) M-1 s-1 at pH 8.3. After the first phase of the reduction, conversion of the red semiquinone to the blue semiquinone was observed in the presence of high concentration of benzoate. This process obeyed first-order kinetics, and the rate increased with an increase of the concentration of benzoate. In addition, the rate was found to be identical with that of the formation of the complex between benzoate and the red semiquinone of DAAO as measured by a stopped-flow method. This suggests that bound benzoate dissociates after the reduction of the benzoate-DAAO complex by benzoate anion radical and that free benzoate subsequently recombines with the red semiquinone of the enzyme to form the blue semiquinone. |
