Studies of Heme Acquisition by the Iron-Regulated Surface Determinant System in Staphylococcus aureus

The work in this dissertation examines how heme is acquired and transferred across the cell wall by receptors of the Iron-regulated surface determinant (Isd) system in Staphylococcus aureus, a bacterial pathogen that requires iron to survive. The Isd system is composed of nine proteins that collaborate with one another to capture heme-iron from hemoglobin (Hb). Hb and heme are recognized by NEAr Transporter (NEAT) domains within the Isd cell wall-attached proteins. A key step in heme acquisition is the removal of the cofactor from Hb by the IsdH protein, which contains three NEAT domains. Using a variety of biochemical techniques, it was discovered that the NEAT domains within IsdH exhibit distinct functions. The first two NEAT domains tether Hb close to the bacterial cell surface, thereby enabling the third NEAT domain to efficiently capture its heme. The IsdC protein is the primary focus of the research described in this dissertation. It is a key intermediate in heme acquisition as it is poised to receive heme from upstream surface-exposed hemoreceptors and selectively deliver the molecule to the downstream heme transporter protein complex. To understand how IsdC recognizes heme, the NMR solution structure of the IsdC NEAT domain (IsdC N) bound to a heme analog was determined, revealing that the metal in protoporphyrin is coordinated in an atypical manner through a tyrosine residue. NMR dynamics studies indicate that IsdCN adaptively recognizes the heme by altering the structure and mobility of a hydrophobic pocket. The central step in heme transfer across the bacterial cell wall is the delivery of heme by IsdA to IsdC. To gain insight into this transfer process, newly developed NMR paramagnetic relaxation enhancement (PRE) methods and targeted amino acid mutagenesis were exploited. These methods reveal that IsdA and IsdC transiently associate with one another in the presence of protoporphyrin via a "handclasp" complex and demonstrate that transient, but stereospecific complexes transfer heme after it is captured from Hb. Mutations in IsdA that target the handclasp interface disrupt heme transfer to IsdC and suggests that small molecules which disrupt the transfer complex can serve to limit microbial access to iron.