Purification and Properties of p-Hydroxybenzoate Hydroxylase from Pseudomonas fluorescens

Abstract

The inducible p-hydroxybenzoate hydroxylase of Pseudomonas fluorescens which catalyzes the conversion of p-hydroxybenzoate to protocatechuate has been obtained in crystalline form as an electrophoretically homogenous protein which is very stable in the absence of added cofactors. The molecular weight is estimated to be 65,000. The enzyme contains 1 mole of flavin adenine dinucleotide per mole of protein. In the presence of p-hydroxybenzoate the enzyme-bound flavin is rapidly reduced by TPNH, but not by DPNH or reduced acetyl pyridine nucleotide. Under both anaerobic and aerobic conditions the enzyme can catalyze the rapid reduction of ferricyanide by TPNH. This rapid reaction requires the presence of the aromatic substrate. The absorption spectrum of the enzyme in the visible region is perturbed and the fluorescence of the enzymebound flavin is quenched in the presence of p-hydroxybenzoate. The binding of p-hydroxybenzoate to p-hydroxybenzoate hydroxylase follows 1:1 binding at concentrations of p-hydroxybenzoate less than 3 x 10-4 m . At higher concentrations, deviations from 1:1 binding are observed which correspond to the excess substrate inhibition observed in steady state kinetic analysis. A substrate analogue, 6-hydroxynicotinate, activates the enzyme with respect to reduction of the enzyme-bound flavin by TPNH, but is not hydroxylated during the subsequent reoxidation by molecular oxygen. During anaerobic reduction of the enzyme in the presence of p-hydroxybenzoate, two transient spectral intermediates are observable. These intermediates also occur during catalytic turnover. The intermediates are generated in sequential fashion. No electron paramagnetic resonance signals are associated with the transient spectral intermediates. The affinity of p-hydroxybenzoate hydroxylase for TPNH is enhanced at least 9-fold by p-hydroxybenzoate. The first order rate constant for the reduction of the enzyme-bound flavin by bound TPNH (i.e. kred) is increased 40,000-fold by p-hydroxybenzoate.