Why Is the Oxidation State
of Iron Crucial for the
Activity of Heme-Dependent Aldoxime Dehydratase? A QM/MM Study
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Abstract
Aldoxime dehydratase is a heme-containing enzyme that
utilizes
the ferrous rather than the ferric ion to catalyze the synthesis of
nitriles by dehydration of the substrate. We report a theoretical
study of this enzyme aimed at elucidating its catalytic mechanism
and understanding this oxidation state preference (Fe<sup>2+</sup> versus Fe<sup>3+</sup>). The uncatalyzed dehydration reaction was
modeled by including three and four water molecules to assist in the
proton transfer, but the computed barriers were very high at both
the DFT (B3LYP) and coupled cluster CCSD(T) levels. The enzymatic
dehydration of <i>Z</i>-acetaldoxime was explored through
QM/MM calculation using two different QM regions and covering all
three possible spin states. The reaction starts by substrate coordination
to Fe<sup>2+</sup> via its nitrogen atom to form a six-coordinated
singlet reactant complex. The ferrous heme catalyzes the N–O
bond cleavage by transferring one electron to the antibond in the
singlet state, while His320 functions as a general acid to deliver
a proton to the leaving hydroxide, thus facilitating its departure.
The key intermediate is identified as an Fe<sup>III</sup>(CH<sub>3</sub>CHN<sup>•</sup>) species (triplet or open-shell singlet),
with the closed-shell singlet Fe<sup>II</sup>(CH<sub>3</sub>CHN<sup>+</sup>) being about 6 kcal/mol higher. Subsequently, the same His320
residue abstracts the α-proton, coupled with electron transfer
back to the iron center. Both steps are calculated to have feasible
barriers (14–15 kcal/mol), in agreement with experimental kinetic
studies. For the same mode of substrate coordination, the ferric heme
does not catalyze the N–O bond cleavage, because the reaction
is endothermic by about 40 kcal/mol, mainly due to the energetic penalty
for oxidizing the ferric heme. The alternative binding option, in
which the anionic aldoxime coordinates to the ferric ion via its oxyanion,
also results in a high barrier (around 30 kcal/mol), mainly because
of the large endothermicity associated with the generation of a suitable
base (neutral His320) for proton abstraction