Intradiol dioxygenases (IDOs) contain non-heme mononuclear Fe(III) and catalyze the cleavage of the aromatic ring of catecholic substrates between the vicinal OH functions with incorporation of both oxygen atoms from O2. There is no experimental evidence for metal redox cycling during catalysis. As such, IDOs are one of only two oxygenase families that utilize an oxidized metal center for catalysis, implying a unique catalytic mechanism. Structural studies show that the substrate binds as a chelate to the iron. We have proposed that O2 then binds in a concerted process leading to an alkylperoxo intermediate. A Criegee rearrangement of this species would lead to ring cleavage and oxygen incorporation. Here, we report the first structural characterization of an oxygenated intermediate in an IDO. The reaction was initiated by adding O2 to a single crystal of protocatechuate 3,4-dioxygenase (3,4-PCD) complexed with a slow alternative substrate. Rapid freezing of the crystal after a short incubation trapped the intermediate in all three of the active sites in the asymmetric unit, allowing us to obtain a 1.8 � resolution structure. Our experiments show that this intermediate is catalytically relevant and is fully converted into cleaved product by the crystals after further incubation. The roles of several conserved active site residues in promoting formation and stabilization of the intermediate are revealed by the structure. This work has major implications for the next generation of inorganic model compounds designed to mimic intradiol chemistry as well as the engineering of IDOs to fully harness their potential in industrial synthesis and remediation. Supported by NIH GM24689.

Reactive oxygen intermediate trapped in a single crystal of an intradiol dioxygenase

248th National Meeting (2014)

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