Substrate-Triggered Addition of Dioxygen to the Diferrous Cofactor of Aldehyde-Deformylating Oxygenase to Form a Diferric-Peroxide Intermediate

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_<i>n</i> fatty aldehydes to formate and the corresponding C_<i>n</i>–1 alkanes or alkenes, respectively. This unusual, apparently redox-neutral transformation actually requires four electrons per turnover to reduce the O₂ cosubstrate to the oxidation state of water and incorporates one O-atom from O₂ 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 O₂ to form a colored intermediate with spectroscopic properties suggestive of a Fe₂^III/III complex with a bound peroxide. Its Mössbauer spectra reveal that the intermediate possesses an antiferromagnetically (AF) coupled Fe₂^III/III center with resolved subsites. The intermediate is long-lived in the absence of a reducing system, decaying slowly (<i>t</i>_1/2 ∼ 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 (^MeOPMS) 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 O₂ moiety to form a Fe₂^III/III-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