Kinetic Mechanisms of the Oxygenase from a Two-component Enzyme, p-Hydroxyphenylacetate

p-Hydroxyphenylacetate hydroxylase (HPAH) from Acinetobacter baumannii catalyzes the hydroxylation of p-hydroxyphenylacetate (HPA) to form 3,4-dihydroxyphenylacetate (DHPA). The enzyme sys-tem is composed of two proteins: an FMN reductase (C1) and an oxy-genase 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 0.2M), but less tightly to oxidized FMN (Kd, 250 50 M). The complex of C2-FMNH reacted with oxygen to form C(4a)-hydroperoxy-FMN at 1.1 0.1 106 M1 s1, whereas the C2-FMNH-HPA complex reacted with oxygen to form C(4a)-hydroperoxy-FMN-HPAmore slowly (k 4.8 0.2 104 M1 s1). The kineticmechanism of C2 was shown to be a preferential ran-dom order type, in which HPA or oxygen can initially bind to the C2-FMNH complex, but the preferred pathwas oxygen reactingwith C2-FMNH to form the C(4a)-hydroperoxy-FMN intermediate prio