title: 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(n) fatty aldehydes to formate and the corresponding C(n-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 (Np) 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 (t(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 ((MeO)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 diiron 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.
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Maria E
authors.initials: ME
authors.lastname: Pandelia
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Ning
authors.initials: N
authors.lastname: Li
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Hanne
authors.initials: H
authors.lastname: Nørgaard
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Douglas M
authors.initials: DM
authors.lastname: Warui
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Lauren J
authors.initials: LJ
authors.lastname: Rajakovich
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Wei-Chen
authors.initials: WC
authors.lastname: Chang
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802.|Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Squire J
authors.initials: SJ
authors.lastname: Booker
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802.|Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Carsten
authors.initials: C
authors.lastname: Krebs
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802.|Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: J Martin
authors.initials: JM
authors.lastname: Bollinger
mesh_terms: D000446:Aldehyde-Lyases / Q000737:chemistry / Q000378:metabolism*; D000447:Aldehydes / Q000737:chemistry / Q000378:metabolism*; D005561:Formates / Q000737:chemistry / Q000378:metabolism; D008968:Molecular Conformation; D046937:Nostoc / Q000201:enzymology; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D010545:Peroxides / Q000737:chemistry / Q000378:metabolism*; D015204:Spectroscopy, Mossbauer; D013379:Substrate Specificity
chemical_list: D000447:Aldehydes; D005561:Formates; D010545:Peroxides; C030544:formic acid; D000446:Aldehyde-Lyases; C043439:aldehyde decarbonylase; D010100:Oxygen