3 research outputs found
Mechanism of Oxygen Activation in a Flavin-Dependent Monooxygenase: A Nearly Barrierless Formation of C4a-Hydroperoxyflavin via Proton-Coupled Electron Transfer
Understanding how flavin-dependent
enzymes activate oxygen for
their oxidation and oxygenation reactions is one of the most challenging
issues in flavoenzymology. Density functional calculations and transient
kinetics were performed to investigate the mechanism of oxygen activation
in the oxygenase component (C<sub>2</sub>) of <i>p</i>-hydroxyphenylacetate
3-hydroxylase (HPAH). We found that the protonation of dioxygen by
His396 via a proton-coupled electron transfer mechanism is the key
step in the formation of the triplet diradical complex of flavin semiquinone
and <sup>•</sup>OOH. This complex undergoes intersystem crossing
to form the open-shell singlet diradical complex before it forms the
closed-shell singlet C4a-hydroperoxyflavin intermediate (C4aOOH).
Notably, density functional calculations indicated that the formation
of C4aOOH is nearly barrierless, possibly facilitated by the active
site arrangement in which His396 positions the proximal oxygen of
the <sup>•</sup>OOH in an optimum position to directly attack
the C4a atom of the isoalloxazine ring. The nearly barrierless formation
of C4aOOH agrees well with the experimental results; based on transient
kinetics and Eyring plot analyses, the enthalpy of activation for
the formation of C4aOOH is only 1.4 kcal/mol and the formation of
C4aOOH by C<sub>2</sub> is fast (∼10<sup>6</sup> M<sup>–1</sup> s<sup>–1</sup> at 4 °C). The calculations identified
Ser171 as the key residue that stabilizes C4aOOH by accepting a hydrogen
bond from the HÂ(N5) of the isoalloxazine ring. Both Ser171 and Trp112
facilitate H<sub>2</sub>O<sub>2</sub> elimination by donating hydrogen
bonds to the proximal oxygen of the OOH moiety during the proton transfer.
According to our combined theoretical and experimental studies, the
existence of a positively charged general acid at the position optimized
for facilitating the proton-coupled electron transfer has emerged
as an important catalytic feature for the oxygen activation process
in flavin-dependent enzymes
Control of C4a-Hydroperoxyflavin Protonation in the Oxygenase Component of <i>p</i>‑Hydroxyphenylacetate-3-hydroxylase
The
protonation status of the peroxide moiety in C4a-(hydro)Âperoxyflavin
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
<i>p</i>‑Hydroxyphenylacetate 3‑Hydroxylase as a Biocatalyst for the Synthesis of Trihydroxyphenolic Acids
Trihydroxyphenolic acids such as
3,4,5-trihydroxycinnamic acid (3,4,5-THCA) <b>4c</b> and 2-(3,4,5-trihydroxyphenyl)Âacetic
acid (3,4,5-THPA) <b>2c</b> are strong antioxidants that are
potentially useful as medicinal agents. Our results show that <i>p</i>-hydroxyphenylacetate (HPA) 3-hydroxylase (HPAH) from <i>Acinetobacter baumannii</i> can catalyze the syntheses of 3,4,5-THPA <b>2c</b> and 3,4,5-THCA <b>4c</b> from 4-HPA <b>2a</b> and <i>p</i>-coumaric acid <b>4a</b>, respectively.
The wild-type HPAH can convert 4-HPA <b>2a</b> completely into
3,4,5-THPA <b>2c</b> within 100 min (total turnover number (TTN)
of 100). However, the wild-type enzyme cannot efficiently synthesize
3,4,5-THCA <b>4c</b>. To improve the efficiency, the oxygenase
component of HPAH (C<sub>2</sub>) was rationally engineered in order
to maximize the conversion of <i>p</i>-coumaric acid <b>4a</b> to 3,4,5-THCA <b>4c</b>. Results from site-directed
mutagenesis studies showed that Y398S is significantly more effective
than the wild-type enzyme for the synthesis of 3,4,5-THCA <b>4c</b>; it can catalyze the complete bioconversion of <i>p</i>-coumaric acid <b>4a</b> to 3,4,5-THCA <b>4c</b> within
180 min (TTN ∼ 23 at 180 min). The yield and stability of 3,4,5-THPA <b>2c</b> and 3,4,5-THCA <b>4c</b> were significantly improved
in the presence of ascorbic acid. Thermostability studies showed that
the wild-type C<sub>2</sub> was very stable and remained active after
incubation at 30, 35, and 40 °C for 24 h. Y398S was moderately
stable because its activity was retained for 24 h at 30 °C and
for 15 h at 35 °C. Transient kinetic studies using stopped-flow
spectrophotometry indicated that the key improvement in the reaction
of Y398S with <i>p</i>-coumaric acid <b>4a</b> lies
within the protein–ligand interaction. Y398S binds to <i>p</i>-coumaric acid <b>4a</b> with higher affinity than
the wild-type enzyme, resulting in a shift in equilibrium toward favoring
the productive coupling path instead of the path leading to wasteful
flavin oxidation