P-Hydroxybenzoate Hydroxylase from Pseudomonas fluorescens: REACTIVITY WITH OXYGEN

Abstract

Abstract p-Hydroxybenzoate hydroxylase, a flavoprotein isolated from Pseudomonas fluorescens, was anaerobically reduced and its reoxidation reactions with O2 studied with a stopped flow spectrophotometer. All effectors of this enzyme which stimulate its TPNH oxidase activity were found also to affect its reactivity with O2. Both the rates of reaction and the binding constant for O2 were enhanced by the effectors. During the reoxidation of the enzyme complexed with the substrate effector, p-hydroxybenzoate, a rapidly formed oxygenated-intermediate is observed. This intermediate is analogous to the oxygenated intermediate previously seen in the presence of 2,4-dihydroxybenzoate, another substrate effector (Spector, T., and Massey, V. (1972) J. Biol. Chem. 247, 5632–5636). It is believed that these intermediates are a hydroperoxide form of the flavin prosthetic group and are catalytic species that are produced prior to hydroxylation. When the reduced enzyme was reoxidized in its uncomplexed form, or complexed with a nonsubstrate effector (i.e. a compound which stimulates the TPNH oxidase activity but is not hydroxylated), no spectral intermediates were detected.