The hydroxylase,
4-hydroxybenzoate 3-monooxygenase, proceeds through a catalytic process that begins with the entrance of
NADPH and 4-hydroxybenzoate (the native
substrate) into the active site of the enzyme. This results in formation of an enzyme-flavin-substrate-NADPH complex, after which the flavin cofactor,
FAD, is reduced by NADPH. NADP+ is lost and O2 enters into the complex, followed by oxidation of the flavin to form a hydroperoxide, which acts as the hydroxide transfer reagent. It is important to note that while the group transferred is referred to as hydroxide it is formally an OH+ group. This hydroxide is transferred to the substrate from the hydroperoxide flavin, flavin-C4a-hydroperoxide, via an
electrophilic aromatic substitution-type reaction. Finally, the product exits from the complex and the hydroxy-flavin is dehydrated, regenerating FAD and allowing the process to repeat. Reduction of the flavin generates a negatively charged species, FADH−, which is attracted to the positively charged active site. This attraction shifts the flavin back to the “closed” conformation, isolating it from the solvent environment. This isolation provides an optimal environment and position for O2 to hydroxylate the substrate. The oxygen binds to FADH− via a
single electron transfer, which is the rate-limiting step of the reaction. This forms an FAD radical and flavin hydroperoxide. Reaction between these generates C4a-peroxyflavin, which is quickly protonated to form flavin-C4a-hydroperoxide.
Tautomerization leads to the formation of 3,4-dihydoxybenzoate. The final step in the mechanism is dissociation of the product and water from FAD, causing the flavin to return to the open conformation. == References ==