Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, poses a great threat to human health. With the emergence of drug resistant Mtb strains, new therapeutics are desperately needed. As iron is critical to the growth and survival of Mtb, the mechanisms through which Mtb acquires host iron pose attractive therapeutic targets. Mtb scavenges iron from the host via Mtb siderophores, carboxymycobactin and mycobactin, and heme uptake. Heme uptake culminates in degradation by the cytosolic protein MhuD, a noncanonical heme degrading enzyme in the IsdG family, in Mtb. In a recent study, a product-bound structure of the MhuD-R26S mutant with ⍺-biliverdin was determined and revealed the formation of a novel secondary structural element, ⍺3, stabilized by the 75HisXXXR79 motif. In Chapter 2 I investigate the biological relevance of this motif by examining the heme binding and degradation of a MhuD R79S variant. I also determined that IsdI, another IsdG-family protein, binds with high affinity to the β- and δ-biliverdin isomers which will enable structural studies of product-bound IsdI. The fate of the heme breakdown product generated by MhuD, mycobilin, remained unknown. In Chapter 3, I demonstrate that Rv2074 is a novel mycobilin reductase and determined the chemical structure of the novel reduced mycobilin product, coined “mycorubin,” utilizing tandem mass spectrometry. Little is known regarding the transport of siderophores across the periplasm and cell-wall environment, but the Mtb periplasmic binding proteins FecB and FecB2 have been implicated in host iron acquisition. In Chapter 4 in vitro ligand binding experiments and structural comparisons for FecB and FecB2 were performed, revealing that both FecB and FecB2 bind heme, while only FecB binds Fe-cMB. Subsequent structure-guided mutagenesis of the FecB ligand binding site identified a single glutamate residue—Glu339—that significantly contributes to Fe-cMB binding. In Chapter 5, a role for FecB in the Mtb siderophore-mediated iron acquisition pathway was corroborated by Mycobacterium smegmatis pull-down experiments, which revealed interactions between FecB and known members of the mycobacterial siderophore export and import machinery, like MmpS5, a protein involved in apo-siderophore efflux. Finally, the FecB interacting partner MmpS5 was confirmed in Mtb by co-immunoprecipitation.