Kinetic mechanisms of the oxygenase from a two-component enzyme, p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii

Abstract

p-Hydroxyphenylacetate hydroxylase (HPAH) from Acinetobacter baumannii catalyzes the hydroxylation of p-hydroxyphenylacetate (HPA) to form 3,4-dihydroxyphenylacetate (DHPA). The enzyme system is composed of two proteins: an FMN reductase (C1) and an oxygenase that uses FMNH- (C2). We report detailed transient kinetics studies at 4 degrees C of the reaction mechanism of C2.C2 binds rapidly and tightly to reduced FMN (Kd, 1.2 +/- 0.2 microm), but less tightly to oxidized FMN (Kd, 250 +/- 50 microm). The complex of C -FMNH-2 reacted with oxygen to form C(4a)-hydroperoxy-FMN at 1.1 +/- 0.1 x 10(6) m(-1) s(-1), whereas the C -FMNH-2 -HPA complex reacted with oxygen to form C(4a)-hydroperoxy-FMN-HPA more slowly (k = 4.8 +/- 0.2 x 10(4) m(-1) s(-1)). The kinetic mechanism of C2 was shown to be a preferential random order type, in which HPA or oxygen can initially bind to the C -FMNH-2 complex, but the preferred path was oxygen reacting with C -FMNH-2 to form the C(4a)-hydroperoxy-FMN intermediate prior to HPA binding. Hydroxylation occurs from the ternary complex with a rate constant of 20 s(-1) to form the C2-C(4a)-hydroxy-FMN-DHPA complex. At high HPA concentrations (>0.5 mm), HPA formed a dead end complex with the C2-C(4a)-hydroxy-FMN intermediate (similar to single component flavoprotein hydroxylases), thus inhibiting the bound flavin from returning to the oxidized form. When FADH- was used, C(4a)-hydroperoxy-FAD, C(4a)-hydroxy-FAD, and product were formed at rates similar to those with FMNH-. Thus, C2 has the unusual ability to use both common flavin cofactors in catalysis.