Decay of the 4a-hydroxy-FAD intermediate of phenol hydroxylase.

The oxidative half-reaction of phenol hydroxylase involves the formation of three spectrally distinct intermediates (Detmer, K.M., and Massey, V. (1985) J. Biol. Chem. 260, 5998-6005). Addition of an aerobic NADPH-regenerating system, phenol, and azide quantitatively converted oxidized enzyme to the third intermediate, a 4a-hydroxy-FAD species (Detmer, K.M., and Massey, V. (1984) J. Biol. Chem. 259, 11265-11272). This intermediate was isolated in the presence of azide and a wide variety of phenolic ligands. Decay rates were followed for the dehydration of 4a-hydroxy-FAD enzyme resulting in the original oxidized form. Deviation from the rate observed in the absence of phenolic ligands was presumed to be indicative of a binding interaction. Several phenols displayed further stabilization of the 4a-hydroxyflavin species. These ligands exhibited saturation kinetics with respect to the decay half-lives, consistent with a mechanistic model in which both free and bound 4a-hydroxy-FAD enzyme may be directly dehydrated to produce the oxidized species. The lack of stabilization by catechol, the natural product, suggests that product is released from the enzyme during turnover by the time that this intermediate is formed. A pH profile, generated for the decay rates in the absence and presence of phenolic ligand, suggests both acid and base catalysis by hydronium ion and hydroxide, respectively.