Diverse substituted phenols can be hydroxylated by flavin-dependent mono-oxygenases in a reaction that can serve as the first step of degradation or transformation. Both single and two-component flavin-dependent monooxygenases are believed to activate substrate for electrophilic aromatic substitution by deprotonating it to the phenolate form. We report experimental tests of this long-standing presumption, using 19F-NMR in conjunction with fluorinated substrate analogs. The single-component monooxygenase 3-hydroxybenzoate-6-hydroxylase (3HB6H) was found to depress the pKa of the substrate analog 4-fluoro-3-hydroxybenzoate (4F3HB) from 8.7 ± 0.03 to 7.1 ± 0.2 as part of binding, consistent with proposed mechanisms, however the two-component mono-oxygenase p-hydroxyphenylacetate 3-hydroxylase (HPAH) proved more complicated. When bound to the C2 component of HPAH, the substrate analog 3-fluoro-4-hydroxyphenylacetate (3F4HPA) responds to a pKa of 8.7 ± 0.1 distinct from that of deprotonation of free 3F4HPA and the dissociation constant decreases upon deprotonation of 3F4HPA with a pKa of 6.6 ± 0.1 when the enzyme is protonated. Mutation of the conserved His120 to Asn and Arg weakened binding of 3F4HPA confirming that His120 forms an important interaction with substrate. Additional experiments are needed to elucidate interactions between multiple ionization events in HPAH's active site and isolate the contributions of His120. Thus in both enzymes we confirm that binding to the hydroxylase component favors the phenolate form of substrate and the 9 kJ/mol and 15 kJ/mol magnitudes of the effect for 3HB6H and HPAH-C2, respectively, are consistent with pKa tuning by one or more H-bonding interactions.

Substrate activation by tuning of pKas in flavin-dependent aromatic hydroxylases

2019 Fall National Meeting

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