Control of C4a-Hydroperoxyflavin Protonation in the
Oxygenase Component of <i>p</i>‑Hydroxyphenylacetate-3-hydroxylase
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Abstract
The
protonation status of the peroxide moiety in C4a-(hydro)peroxyflavin
of <i>p</i>-hydroxyphenylacetate-3-hydroxylase can be directly
monitored using transient kinetics. The p<i>K</i><sub>a</sub> for the wild-type (WT) enzyme is 9.8 ± 0.2, while the values
for the H396N, H396V, and H396A variants are 9.3 ± 0.1, 7.3 ±
0.2, and 7.1 ± 0.2, respectively. The hydroxylation efficiency
of these mutants is lower than that of the WT enzyme. Solvent kinetic
isotope effect studies indicate that proton transfer is not the rate-limiting
step in the formation of C4a-OOH. All data suggest that His396 may
act as an instantaneous proton provider for the proton-coupled electron
transfer that occurs before the transition state of C4a-OOH formation
