Abstract
Methanol oxidase from Hansenula polymorpha contains five "red" flavin semiquinones and two oxidized flavins per octamer. Addition of substrate results in the reduction of the two oxidized flavins but does not affect the flavin semiquinones. Enhanced water proton relaxation rates indicate that the unpaired electron of the flavin semiquinones is accessible to the solvent and this accessibility is significantly decreased upon binding of the suicide inhibitor cyclopropanol. In the native enzyme, the semiquinones are not oxidizable by air. All flavins were resolved from the enzyme, and holoenzyme was reconstituted by addition of oxidized flavin. The reconstituted enzyme was catalytically active. The specific activity was 50% that of the original enzyme. It was concluded that the semiquinone is not required for the oxidation of methanol, although it may be present at an otherwise intact site.