Redox potential-pH properties of the flavoprotein L-amino-acid oxidase

The redox potential-pH characteristics of the enzyme L-amino-acid oxidase (L-amino-acid: oxygen oxidoreductase (deaminating), EC 1.4.3.2) have been measured in the pH range 6.2 to 8.3 at 4 degrees C. All potentials are reported versus the standard hydrogen electrode. Consistent with the protonation states proposed for the anionic red semiquinone and anionic dihydroquinone forms of the flavin coenzyme, the first electron potential is independent of pH (-0.056 +/- 0.006 V), while the second electron transfer potential is pH-dependent, exhibiting a 0.060 V/pH unit slope. At all pH values investigated, the percentage of semiquinone species observed matches closely that calculated from measured potential separations. The semiquinone species is thermodynamically stable, as indicated by formation of semiquinone, similarity of redox potentials in oxidative and reductive directions, and by the slope of Nernst plots.