The roles of Asn7 and Trp67 in tuning the ferryl haem form of Staphylococcus aureus IsdG

IsdG is a member of the Staphylococcus aureus iron-regulated surface determinant system that degrades haem scavanged from a human host to a mixture of staphylobilin and biliverdin; the rate-limiting step of this enzyme is the rearrangment of a ferric–peroxohaem species to a hydroxylated ferryl=oxohaem speices. In this work, the role of conserved Asn and Trp residues in catalysing this reaction are interrogated by preparing and characterizing ferryl haem analogues for the hydroxylated ferryl=oxohaem speices in N7A and W67F IsdG. The ferryl haem form of N7A IsdG had an extremely short lifetime, and could not be detected using scanning UV/Vis absorption (Abs) spectroscopy. In contrast, the ferryl haem form of W67F IsdG could be observed with UV/Vis Abs spectroscopy, but the lifetime of the ferryl haem form of W67F IsdG (110 s) was less than half that reported for wild-type (WT) enzyme (250 s). Magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) characterization of the reaction mixture between ferric heme-bound N7A IsdG and meta-chloroperoxybenzoic acid (mCPBA) did not yield any evidence for a ferryl haem species, further suggesting that the lifetime of the ferryl haem form of N7A IsdG is extremely short. Similarly, clear evidence for a ferryl haem form of IsdG was not obtained upon MCD characterisation of the reaction mixture between the ferric haem form of W67F IsdG and mCPBA. However, EPR characterization of the W67F IsdG reaction provided clear evidence for the presence of a compound I-like ferryl haem species distinct from the compound ES-like ferryl haem reported for WT enzyme. The lifetime measurments revealed that both Asn7 and Trp67 have important roles in stabilizing the ferryl haem form of IsdG, with Asn7 making a more significant contribution. Furthermore, the correlation between organic radical location in the ferryl haem form of IsdG and the ratio of staphylobilin to biliverdin generated by the enzyme suggests that this organic radical plays a major role in determining the product selectivity of IsdG.