Stabilization of the Ferryl═Oxoheme Form of <i>Staphylococcus aureus</i> IsdG by Electron Transfer from a Second-Sphere Tryptophan

The ferryl heme forms of <i>Staphylococcus aureus</i> IsdG and IsdI have novel UV/vis absorption spectra that are distinct from those of the three forms of ferryl heme typically found in biological systems: compound I, compound II, and compound ES. In this work, the ferryl heme form of IsdG was characterized because it is an analogue for the immediate product of enzyme-catalyzed heme hydroxylation. The ferryl heme 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 heme 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 heme. 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, multiscale quantum mechanics/molecular mechanics and time-dependent density functional theory strongly suggest that the ferryl heme form of IsdG has a ruffled porphyrin ligand and an oxo ligand. Thus, the ferryl heme 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.