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

• Main Authors: Nantidaporn Ruangchan, Chanakan Tongsook, Jeerus Sucharitakul, Pimchai Chaiyen
• Other Authors: Mahidol University
• Format: Article
• Published: 2018
• Subjects: Biochemistry, Genetics and Molecular Biology; Medicine
• Online Access: https://repository.li.mahidol.ac.th/handle/123456789/11607

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. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.