Staphylococcus aureus IsdG is the terminal enzyme in the heme iron acquisition pathway wheby this human pathogen acquires iron from its host during infection. Similar to eukaryotic heme oxygenases, IsdG converts a ferric–peroxoheme intermediate to a meso-hydroxyheme intermediate. However, IsdG lacks the active site features present in eukaryotic heme oxygenases that provide structural control of the oxygenation reaction. Previous work has shown that there is a hydrogen bond between the catalytically-essential second-sphere Asn7 residue and the α-atom of the distal ligand to heme iron, and density functional theory calculations have suggested that this second-sphere interaction alters the electronic structure of the porphyrin ligand in a manner that promotes meso carbon hydroxylation. Here, I will present how we have selectively 13C-labeled the heme meso carbons in order to test this computational prediction using 13C nuclear magnetic resonance spectroscopy. We have observed a 10 ppm shift of the average heme meso 13C resonance upon replacing Asn7 with an Ala, which is consistent with a significant increase in spin density at the heme meso carbons upon introduction of an Asn7…distal ligand hydrogen bond. As a result, a second-sphere interaction between Asn7 and the heme iron distal ligand significantly increases the electrophilicity of the heme meso carbons and promotes substrate hydroxylation. This is a novel heme reaction mechanism of fundamental interest to bioinorganic and synthetic inorganic researchers studying the chemistry of iron tetrapyrroles.

Spectroscopic evidence for electronic control of heme hydroxylation by Staphylococcus aureus IsdG

256th National Meeting (2018)

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