A ferric-peroxo intermediate in the oxidation of heme by IsdI

The canonical heme oxygenases (HO) catalyze heme oxidation via a heme-bound hydroperoxo intermediate that is stabilized by a water cluster at the active site of the enzyme. In contrast, the hydrophobic active site of IsdI, a heme degrading enzyme from Staphylococcus aureus, lacks a water cluster and is expected to oxidize heme by an alternative mechanism. Reaction of the IsdI-heme complex with either H₂O-₂ or mCPBA fails to produce a specific oxidized heme iron intermediate, suggesting that ferric-hydroperoxo or ferryl derivatives of IsdI are not involved in the catalytic mechanism of this enzyme. IsdI lacks a proton donating group in the distal heme pocket, so the possible involvement of a ferric-peroxo intermediate has been evaluated. Density functional theory (DFT) calculations indicate that heme oxidation involving a ferric-peroxo intermediate is energetically accessible, whereas the energy barrier for a reaction involving a ferric-hydroperoxo intermediate is too great in the absence of a proton donor. We propose that IsdI catalyzes heme oxidation through nucleophilic attack by the hemebound peroxo species. This proposal is consistent with our previous demonstration by NMR spectroscopy that heme ruffling increases the susceptibility of the meso-carbon of heme to nucleophilic attack.Medicine, Faculty ofScience, Faculty ofBiochemistry and Molecular Biology, Department ofMicrobiology and Immunology, Department ofReviewedFacultyPostdoctora