Studies on the oxidative half-reaction of p-hydroxyphenylacetate 3-hydroxylase

The oxidative half-reaction of the two-protein enzyme, p-hydroxyphenylacetate 3-hydroxylase from Pseudomonas putida, has been studied by absorbance stopped-flow techniques. The formation of three flavin-oxygen intermediates, the anionic and protonated forms of the flavin hydroperoxide (intermediates I and I) and the hydroxyflavin (intermediate III), was observed during the course of the oxygen reaction with the reduced flavoprotein-coupling protein complex. The flavin hydroperoxide, which is formed in a second-order reaction with oxygen, is in rapid equilibrium with the aromatic substrate, p-hydroxyphenylacetate. Due to this rapid equilibrium, p-hydroxyphenylacetate effectively competes with other ligands, such as p-chlorophenylacetate and p-aminophenylacetate and proceeds through the hydroxylation pathway. Furthermore, dehydration of intermediate III is subjected to severe inhibition in the presence of excess p-hydroxyphenylacetate, similar to the observations made with phenol hydroxylase. A reaction mechanism for the oxidative half-reaction in the presence of the aromatic substrate, p-hydroxyphenylacetate, is proposed.