pH-dependent studies reveal an efficient hydroxylation mechanism of the oxygenase component of p-hydroxyphenylacetate 3-hydroxylase.

p-Hydroxyphenylacetate (HPA) 3-hydroxylase (HPAH) catalyzes the hydroxylation of HPA at the ortho-position to yield 3,4-dihydroxyphenylacetate. The enzyme is a flavin-dependent two-component monooxygenase that consists of a reductase component and an oxygenase component (C(2)). C(2) catalyzes the hydroxylation of HPA using oxygen and reduced FMN as co-substrates. To date, the effects of pH on the oxygenation of the two-component monooxygenases have never been reported. Here, we report the reaction kinetics of C(2)·FMNH(-) with oxygen at various pH values investigated by stopped-flow and rapid quenched-flow techniques. In the absence of HPA, the rate constant for the formation of C4a-hydroperoxy-FMN (∼1.1 × 10(6) m(-1)s(-1)) was unaffected at pH 6.2-9.9, which indicated that the pK(a) of the enzyme-bound reduced FMN was less than 6.2. The rate constant for the following H(2)O(2) elimination step increased with higher pH, which is consistent with a pK(a) of >9.4. In the presence of HPA, the rate constants for the formation of C4a-hydroperoxy-FMN (∼4.8 × 10(4) m(-1)s(-1)) and the ensuing hydroxylation step (15-17 s(-1)) were not significantly affected by the pH. In contrast, the following steps of C4a-hydroxy-FMN dehydration to form oxidized FMN occurred through two pathways that were dependent on the pH of the reaction. One pathway, dominant at low pH, allowed the detection of a C4a-hydroxy-FMN intermediate, whereas the pathway dominant at high pH produced oxidized FMN without an apparent accumulation of the intermediate. However, both pathways efficiently catalyzed hydroxylation without generating significant amounts of wasteful H(2)O(2) at pH 6.2-9.9. The decreased accumulation of the intermediate at higher pH was due to the greater rates of C4a-hydroxy-FMN decay caused by the abolishment of substrate inhibition in the dehydration step at high pH.

Structure of the monooxygenase component of a two-component flavoprotein monooxygenase.

p-Hydroxyphenylacetate hydroxylase from Acinetobacter baumannii is a two-component system consisting of a NADH-dependent FMN reductase and a monooxygenase (C2) that uses reduced FMN as substrate. The crystal structures of C2 in the ligand-free and substrate-bound forms reveal a preorganized pocket that binds reduced FMN without large conformational changes. The Phe-266 side chain swings out to provide the space for binding p-hydroxyphenylacetate that is oriented orthogonal to the flavin ring. The geometry of the substrate-binding site of C2 is significantly different from that of p-hydroxybenzoate hydroxylase, a single-component flavoenzyme that catalyzes a similar reaction. The C2 overall structure resembles the folding of medium-chain acyl-CoA dehydrogenase. An outstanding feature in the C2 structure is a cavity located in front of reduced FMN; it has a spherical shape with a 1.9-A radius and a 29-A3 volume and is interposed between the flavin C4a atom and the substrate atom to be hydroxylated. The shape and position of this cavity are perfectly fit for housing the oxygen atoms of the flavin C4a-hydroperoxide intermediate that is formed upon reaction of the C2-bound reduced flavin with molecular oxygen. The side chain of His-396 is predicted to act as a hydrogen-bond donor to the oxygen atoms of the intermediate. This architecture promotes the nucleophilic attack of the substrate onto the terminal oxygen of the hydroperoxyflavin. Comparative analysis with the structures of other flavoenzymes indicates that a distinctive feature of monooxygenases is the presence of specific cavities that encapsulate and stabilize the crucial hydroperoxyflavin intermediate.