Tuning of pK_a values activates substrates in flavin-dependent aromatic hydroxylases

Hydroxylation of substituted phenols by flavin-dependent monooxygenases is the first step of their biotransformation in various microorganisms. The reaction is thought to proceed via electrophilic aromatic substitution, catalyzed by enzymatic deprotonation of substrate, in single-component hydroxylases that use flavin as a cofactor (group A). However, two-component hydroxylases (group D), which use reduced flavin as a co-substrate, are less amenable to spectroscopic investigation. Herein, we employed ¹⁹F NMR in conjunction with fluorinated substrate analogs to directly measure pK_a values and to monitor protein events in hydroxylase active sites. We found that the single-component monooxygenase 3-hydroxybenzoate 6-hy-droxylase (3HB6H) depresses the pK_a of the bound substrate analog 4-fluoro-3-hydroxybenzoate (4F3HB) by 1.6 pH units, consistent with previously proposed mechanisms. ¹⁹F NMR was applied anaerobically to the two-component monooxygenase 4-hydroxyphenylacetate 3-hydroxylase (HPAH), revealing depression of the pK_a of 3-fluoro-4-hydroxyphenylacetate by 2.5 pH units upon binding to the C₂ component of HPAH. ¹⁹F NMR also revealed a pK_a of 8.7 ± 0.05 that we attributed to an active-site residue involved in deprotonating bound substrate, and assigned to His-120 based on studies of protein variants. Thus, in both types of hydroxylases, we confirmed that binding favors the phenolate form of substrate. The 9 and 14 kJ/mol magnitudes of the effects for 3HB6H and HPAH-C₂, respectively, are consistent with pK_a tuning by one or more H-bonding interactions. Our implementation of ¹⁹F NMR in anaerobic samples is applicable to other two-component flavin-dependent hydroxylases and promises to expand our understanding of their catalytic mechanisms.