Fluoride elimination from substrates in hydroxylation reactions catalyzed by p-hydroxybenzoate hydroxylase

Several fluorinated derivatives of p-hydroxybenzoate were synthesized and examined as substrates in the reaction catalyzed by p-hydroxybenzoate hydroxylase. All the derivatives tested served as substrates, undergoing tightly coupled hydroxylation by molecular oxygen. Hydroxylation of the difluoro and tetrafluoro derivatives liberated stoichiometric amounts of fluoride. Little or no fluoride was released with monofluoro substrates. The defluorination caused higher consumption of NADPH with an overall NADPH to oxygen ratio of 2, in contrast to the ratio of 1 with the physiological substrate and with the monofluoro derivatives. Evidence was obtained strongly suggestive of a quinonoid species as the primary product formed upon oxygenative defluorination. The additional equivalent of NADPH consumed upon fluoride elimination is presumably used in a nonenzymatic reaction with the quinonoid intermediate, resulting in the observed dihydroxy product. Stopped flow studies of the reductive and oxidative half-reactions with tetrafluoro-p hydroxybenzoate substrate were examined. The oxygen half-reaction was analogous to that with p-hydroxybenzoate involving two transient oxygenated flavin intermediates. The decay of the first intermediate, a C(4a)-peroxyflavin, results in rupture of the oxygen-oxygen bond and is rate-determining in overall catalysis. This is in contrast to the reaction with the normal substrate, presumably due to a deactivating effect of the fluorine substituents. The above results are consistent with an oxenoid mechanism of oxygen attack.