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 mono-unsaturated Cn fatty aldehydes to formate and the corresponding Cn-1 alkanes or alkenes, respectively. This unusual, apparently redox-neutral transformation actually requires four electrons per turnover to reduce the O2 co-substrate to the oxidation state of water and incorporates one O-atom from O2 into the formate co-product. We show here that the complex of the diiron(II/II) form of ADO from Nostoc punctiforme (Np) with an aldehyde substrate reacts with O2 to form a colored intermediate with spectroscopic properties suggestive of a Fe2III/III complex with a bound peroxide. Its Mössbauer spectra reveal that the intermediate possesses an antiferromagnetically (AF) coupled Fe2III/III center with resolved sub-sites. The intermediate is long-lived in the absence of a reducing system, decaying slowly (t1/2 ~ 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-methylphenazine (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 diiron cofactor (rather than a homo- or hetero-dinuclear 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 Fe2III/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.

A growing family of O2 activating dinuclear iron enzymes with key catalytic diiron(III)-peroxo intermediates: Biological systems and chemical models

“…Recent stopped flow UV-visible and Mössbauer spectroscopic data support the formation of a peroxy-hemiacetal species [101,102]. Indeed, in these experiments, the formation of a peroxo-Fe III Fe III species was interrupted by the appearance of an absorption band at 450 nm, which was depending on the presence of a substrate.…”

“…9) [98,101]. The catalytic cycle begins with the reduction of the Fe III Fe III species into a Fe II Fe II center capable of performing the reductive activation of dioxygen.…”

Organophosphonate-degrading PhnZ reveals an emerging family of HD domain mixed-valent diiron oxygenases

“…His62, in particular, seems important for proper orientation of the substrate, analogous to Lys127 in MIOX, which is essential for activity (6). With the exception of MIOX, all dinuclear nonheme-iron oxygenases and oxidases studied to date use the fully reduced forms of their cofactors to activate O 2 (32)(33)(34)(35)(36)(37)(38), and the mixedvalent Fe II /Fe III forms are usually only marginally stable in these enzymes (28,39,40). By contrast, MIOX employs the mixedvalent form of its diiron cluster to promote substrate and O 2 activation and accordingly stabilizes this state, allowing its accumulation in 60-70% yield (17 (Fig.…”

Structural Basis for Oxygen Activation at a Heterodinuclear Manganese/Iron Cofactor

“…The protein may well bind its physiological substrate(s) in the reduced state, and the substrate may similarly be only weakly coordinated to the metal ions, so that oxygen activation is possible. Such a scenario is encountered in the distantly related cyanobacterial aldehyde-deformylating oxygenase, where oxygen activation is triggered by substrate binding (28).…”