Substrate-Triggered Formation of a Peroxo-Fe₂(III/III) Intermediate during Fatty Acid Decarboxylation by UndA

Abstract

The iron-dependent oxidase UndA cleaves one C3–H bond and the C1–C2 bond of dodecanoic acid to produce 1-undecene and CO2. A published X-ray crystal structure showed that UndA has a heme-oxygenase-like fold, thus associating it with a structural superfamily that includes known and postulated non-heme diiron proteins, but revealed only a single iron ion in the active site. Mechanisms proposed for initiation of decarboxylation by cleavage of the C3–H bond using a monoiron cofactor to activate O2 necessarily invoked unusual or potentially unfeasible steps. Here we present spectroscopic, crystallographic, and biochemical evidence that the cofactor of Pseudomonas fluorescens Pf-5 UndA is actually a diiron cluster and show that binding of the substrate triggers rapid addition of O2 to the Fe2(II/II) cofactor to produce a transient peroxo-Fe2(III/III) intermediate. The observations of a diiron cofactor and substrate-triggered formation of a peroxo-Fe2(III/III) intermediate suggest a small set of possible mechanisms for O2, C3–H and C1–C2 activation by UndA; these routes obviate the problematic steps of the earlier hypotheses that invoked a single iron.

Structure and assembly of the diiron cofactor in the heme-oxygenase-like domain of theN-nitrosourea-producing enzyme SznF

“…As in Fe 2 (II/II)-SznF, core a3 of CADD, which we presume to have been characterized in its oxidized Fe 2 (III/III) state, is fully a-helical in the published structure. By contrast, UndA shares with SznF the property of cofactor instability and still has not been crystallographically characterized with its diiron cluster intact, despite concerted efforts toward this goal (43). Core a3 in the UndA structure is partially disordered, as in the previously reported structures of SznF (37), and the Asp315 equivalent, Asp198, resides in or near this unmodeled stretch (Fig.…”

“…within the relatively open HDO core, and it was initially suggested that UndA uses the observed monoiron(II) center for O 2 activation (41). A pair of later studies showed, by contrast, that the iron cofactor is dinuclear (42,43). One of these studies characterized a µ-peroxo-Fe 2 (III/III) complex on the reaction pathway (43).…”

“…A pair of later studies showed, by contrast, that the iron cofactor is dinuclear (42,43). One of these studies characterized a µ-peroxo-Fe 2 (III/III) complex on the reaction pathway (43). In that work, concerted efforts to solve a structure with both cofactor subsites fully occupied were unsuccessful, and regions of the protein implicated in providing ligands for the second iron site (core a3) gave weak electron density, indicative of structural disorder.…”

“…S3) (37). The apparent conservation of cofactor instability among the first several functionally assigned proteins in the emerging HDO superfamily (37,38,40,41,43) was taken as evidence that they might function by a novel modus operandi, perhaps utilizing iron more as a labile cosubstrate than as a stably bound, in-situ-recycled cofactor (39). Regardless of the reason, no structure of a catalytically relevant (diferrous or diferric) holoenzyme HDO complex for a functionally assigned member of this emerging superfamily has yet been reported.…”

“…S9). However, the fact that substitution of this residue abolished activity in UndA was taken to imply that it is a cofactor ligand, which would, in turn, suggest that it must also be brought into position during assembly of the O 2 -reactive complex (43). Here, an additional event, fatty acid (substrate) binding, is necessary to produce the reactive state.…”