Kinetic and isotopic studies of the oxidative half-reaction of phenol hydroxylase

Phenol hydroxylase, an FAD-containing monooxygenase, catalyzes the conversion of substituted phenols to the corresponding catechol. Use of metapyrocatechase, capable of dioxygenation of several catechols to give highly absorbing products, permitted determination of the time course of product release from phenol hydroxylase. Product dissociated prior to complete reoxidation of the enzyme, most likely concomitant with formation of the 4a-hydroxyflavin species (intermediate III). Deuterated phenol and thiophenol exhibited no kinetic isotope effect during the oxidative half-reaction. Isotope effects of 1.7 to 3.7 were found with resorcinol for the conversion of the second intermediate to intermediate III. These effects limited the possible models for phenol hydroxylation. An attempt was made to distinguish whether the spectrum of intermediate II is due entirely to that of the flavin moiety of phenol hydroxylase or whether some radical intermediate form involved in the formation of catechol makes a significant visible contribution. Reduced native and 6-hydroxy-FAD phenol hydroxylase were reacted with oxygen and resorcinol in order to provide evidence for the identity of intermediate II.