Substrate-Triggered
Addition of Dioxygen to the Diferrous
Cofactor of Aldehyde-Deformylating Oxygenase to Form a Diferric-Peroxide
Intermediate
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Abstract
Cyanobacterial
aldehyde-deformylating oxygenases (ADOs) belong
to the ferritin-like diiron-carboxylate superfamily of dioxygen-activating
proteins. They catalyze conversion of saturated or monounsaturated
C<sub><i>n</i></sub> fatty aldehydes to formate and the
corresponding C<sub><i>n</i>–1</sub> alkanes or alkenes,
respectively. This unusual, apparently redox-neutral transformation
actually requires four electrons per turnover to reduce the O<sub>2</sub> cosubstrate to the oxidation state of water and incorporates
one O-atom from O<sub>2</sub> into the formate coproduct. We show
here that the complex of the diiron(II/II) form of ADO from Nostoc punctiforme (<i>Np</i>) with an
aldehyde substrate reacts with O<sub>2</sub> to form a colored intermediate
with spectroscopic properties suggestive of a Fe<sub>2</sub><sup>III/III</sup> complex with a bound peroxide. Its Mössbauer spectra reveal
that the intermediate possesses an antiferromagnetically (AF) coupled
Fe<sub>2</sub><sup>III/III</sup> center with resolved subsites. The
intermediate is long-lived in the absence of a reducing system, decaying
slowly (<i>t</i><sub>1/2</sub> ∼ 400 s at 5 °C)
to produce a very modest yield of formate (<0.15 enzyme equivalents),
but reacts rapidly with the fully reduced form of 1-methoxy-5-methylphenazinium
methylsulfate (<sup>MeO</sup>PMS) to yield product, albeit at only
∼50% of the maximum theoretical yield (owing to competition
from one or more unproductive pathway). The results represent the
most definitive evidence to date that ADO can use a <i>diiron</i> cofactor (rather than a homo- or heterodinuclear cluster involving
another transition metal) and provide support for a mechanism involving
attack on the carbonyl of the bound substrate by the reduced O<sub>2</sub> moiety to form a Fe<sub>2</sub><sup>III/III</sup>-peroxyhemiacetal
complex, which undergoes reductive O–O-bond cleavage, leading
to C1–C2 radical fragmentation and formation of the alk(a/e)ne
and formate products