Stabilization of the Ferryl=oxoheme Form of Staphylococcus aureus IsdG by Electron Transfer from a Second-Sphere Tryptophan

The ferryl=oxoheme forms of Staphylococcus aureus IsdG and IsdI have novel UV/Vis absorption spectra that are distinct from those of the three forms of ferryl=oxoheme typically found in biological systems: compound I, compound II, and compound ES. In this work, the ferryl=oxoheme form of IsdG was characterized because it is an analogue for the immediate product of enzyme-catalyzed heme hydroxylation. The ferryl=oxoheme form of IsdG generated following the addition of meta-chloroperoxybenzoic acid to the ferric heme form of IsdG has a half-life of 4.0 ± 0.2 min, which is more than 100 times longer than the half-life for the ferryl=oxoheme form of human heme oxygenase (hHO). Magnetic circular dichroism characterization of the IsdG species yielded spectral data and zero-field splitting parameters consistent with either a compound II- or compound ES-like ferryl=oxoheme. Further characterization of isotopically-enriched samples with electron paramagnetic resonance spectroscopy revealed the presence of a protein-based organic radical, as would be expected for compound ES. Finally, multi-scale quantum mechanics / molecular mechanics and time-dependent density functional theory strongly suggest that the ferryl=oxoheme form of IsdG has a ruffled porphyrin ligand and deprotonated oxo ligand. Thus, the ferryl=oxoheme form of IsdG is assigned to a compound ES-like species with a Trp67-based radical. Electron transfer from Trp67 to porphyrin will stabilize the immediate product of heme hydroxylation and provide a thermodynamic driving force for the reaction. Furthermore, the ability to transfer an electron between Trp67 and the substrate may explain the differential reactivity of meso-hydroxyheme in IsdG and hHO.