P-Hydroxybenzoate Hydroxylase from Pseudomonas fluorescens: EVIDENCE FOR AN OXYGENATED FLAVIN INTERMEDIATE

Abstract

Abstract The slow reaction of 2,4-dihydroxybenzoate (2,4-DOHB) with p-hydroxybenzoate hydroxylase in the presence of TPNH and oxygen, and the absence of auto-inhibition at high concentrations of 2,4-DOHB has facilitated a study of the hydroxylation reaction mechanism of this enzyme. The Vmax (45 min-1) of the over-all reaction and other kinetic constants were determined with steady state kinetics. Stopped flow spectrophotometric studies of the anaerobic reduction of the p-hydroxybenzoate hydroxylase-2,4-DOHB (E-S) complex by TPNH has demonstrated the formation of at least three intermediates during this process. All three species exhibit long wave length absorption bands but lack any detectable free radical EPR signals. Stopped flow studies on the reoxidation of the reduced E-S complex by oxygen have revealed the formation of a spectral intermediate that is believed to be an oxygenated form of the enzyme-bound flavin prosthetic group. A second intermediate tentatively identified as the oxidized enzyme-product complex is also detected during the reoxidation. This species decays at a rate similar to that of the Vmax; it is therefore likely that product dissociation is the over-all rate-limiting step.