Kinetic Mechanisms of the Oxygenase from a Two-component Enzyme, p-Hydroxyphenylacetate 3-Hydroxylase from 'Acinetobacter baumannii'

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°C of the reaction mechanism of C₂. C₂ binds rapidly and tightly to reduced FMN (Kd, 1.2 ± 0.2μM), but less tightly to oxidized FMN (Kd, 250 ± 50μM). The complex of C₂-FMNH⁻ reacted with oxygen to form C(4a)-hydroperoxy-FMN at 1.1 ± 0.1 x 10⁶M⁻¹S⁻¹, whereas the C₂-FMNH⁻-HPA complex reacted with oxygen to form C(4a)-hydroperoxy-FMN-HPA more slowly (k=4.8±0.2 x 10⁴M⁻¹S⁻¹).The kinetic mechanism of C₂was shown to be a preferential random order type, in which HPA or oxygen can initially bind to the C₂-FMNH⁻ complex, but the preferred path was oxygen reacting with C₂-FMNH⁻ to form the C(4a)-hydroperoxy-FMN intermediate prior to HPA binding. Hydroxylation occurs from the ternary complex with a rate constant of 20S⁻¹ to form the C₂-C(4a)-hydroxy-FMN-DHPA complex. At high HPA concentrations (>0.5mM), 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.