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

Abstract

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 species. 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 species in N7A and W67F IsdG. The ferryl haem form of N7A IsdG had an extremely short lifetime, and could only be detected by lowering the temperature to 5 °C. In contrast, the ferryl haem form of W67F IsdG could be observed at room temperature, 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, consistent with the extremely short lifetime of the ferryl haem form of N7A IsdG. Similarly, clear evidence for a ferryl haem form of IsdG was not obtained upon MCD characterization 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 or compound ES-like ferryl haem species. The lifetime measurements 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 lifetime in the ferryl haem form of IsdG and the ratio of staphylobilin to biliverdin generated by the enzyme suggests that this organic radical may play a major role in determining the product selectivity of IsdG.