Time-resolved studies of IsdG identify molecular signposts along the non-canonical heme oxygenase pathway.

IsdGs are heme monooxygenases that break open the tetrapyrrole, releasing the iron and thereby allowing bacteria expressing this protein to use heme as a nutritional iron source. Little is currently known about the mechanism by which IsdGs degrade heme, though the products differ from those generated by canonical heme oxygenases. A synthesis of time resolved techniques including in proteo mass spectrometry and conventional and stopped flow UV/vis was used in conjunction with analytical methods to define the reaction steps mediated by IsdG from Staphylococcus aureus and their time scales. An apparent meso -(hydr)oxyheme (forming with k = 0.6 min(-1), pH 7.4, 10 mM ascorbate, 10 μM IsdG-heme, 22 °C) was identified as a likely common intermediate with the canonical heme oxygenases (HOs). Unlike HOs, this intermediate does not form with added H2O2, nor does it convert to verdoheme and CO. Rather, the next observable intermediates (k = 0.16 min(-1)) were a set of formyl-oxo-bilin isomers, similar to the mycobilin products of the IsdG homolog from Mycobacterium tuberculosis (MhuD). These converted in separate fast and slow phases to β-/δ-staphylobilin isomers and formaldehyde (CH2O). Controlled release of this unusual C1 product may support IsdG's dual role as both an oxygenase and a sensor of heme availability in S. aureus.