Studies on the Mechanism of Phenol Hydroxylase.

Abstract

Phenol hydroxylase (EC 1.14.13.7) is a flavoprotein catalyzing the incorporation of one atom of molecular oxygen into an aromatic ring ortho to the existing hydroxyl group. The enzyme has been studied by steady state and rapid reaction techniques. The enzyme binds the phenolic substrate and NADPH in that order, followed by rapid transfer of electrons from pyridine nucleotide to flavin. In the absence of substrate, the dissociation constant for NADPH binding in several orders of magnitude larger than in the presence of substrate. After NADP('+) dissociates, oxygen reacts with the reduced enzyme/substrate complex to form products and oxidized enzyme. Three intermediates can be detected in the oxidative half reaction by stopped flow spectrophotometry in the presence of monovalent anions. Monovalent anions are inhibitors of phenol hydroxylase, affecting the conversion of the third intermediate to oxidized enzyme. The rate of formation of the first intermediate is dependent on the oxygen concentration. The first and third intermediates have been identified as the flavin C(4a)-hydroperoxide and hydroxide, respectively. The structure of intermediate II is unknown. Oxygen transfer takes place with the conversion of intermediate I to II. A linear free energy plot of the rate of conversion of I to II versus the (sigma)('+) value for a series of meta-substituted phenols gives a straight line with a slope of -0.5, consistent with an electrophilic aromatic substitution reaction. In the oxidative half reaction, the conversion of intermediates I to II and II to III are catalyzed by acid. A kinetic isotope effect of 8.5 has been measured using deuterated resorcinol for the conversion of intermediate II to intermediate III, indicating that product rearomatization is necessary for this conversion. The conversion of intermediate III to oxidized enzyme is base-catalyzed, consistent with this step being dependent on loss of a proton from the flavin N(5)-position, followed by expulsion of the flavin C(4a)-hydroxide. Product release can take place before the conversion of intermediate III to oxidized enzyme. When the native FAD is replaced with FAD chemically modified in the pyrimidine ring, the enzyme is not capable of hydroxylating substrate.