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Background and Objective: ATP-binding cassette (ABC) transporters are prominent drug targets due to their highly efficient trafficking capabilities and their significant physiological and clinical roles. Gaining insight into their biophysical and biomechanistic properties is crucial to maximize their pharmacological potential. Materials and Methods: In this study, we present the biochemical and biophysical characterization, and phylogenetic analysis of the domains of Mycobacterium tuberculosis (M. tuberculosis) ABC transporters: the exporter Rv1348 (IrtA) and the importer system Rv1349-Rv2895c (IrtB-Rv2895c), both involved in siderophore-mediated iron uptake. Results: Our findings reveal that the substrate-binding domain (SBD) of IrtA functions as an active monomer, while Rv2895c, which facilitates the uptake of siderophore-bound iron, exists in a dynamic equilibrium between dimeric and monomeric forms. Furthermore, ATP binding induces the dimerization of the ATPase domains in both IrtA (ATPase I) and IrtB (ATPaseII), but only the ATPase domain of IrtA (ATPase I) is active independently. We also analyzed the stability of substrate binding to the domains of the two transporters across varying temperature and pH ranges, revealing significant shifts in their activity under different conditions. Our study highlights the conformational changes that accompany substrate interaction with the transporter domains, providing insights into the fundamental mechanism required for the translocation of siderophore to the extracytoplasmic milieu by IrtB and, subsequently, import of their ferrated forms by the IrtB-Rv2895c complex. Phylogenetic analyses based on ATPase domains reveal that IrtA shares features with both archaeal and eukaryotic transporters, while IrtB is unique to mycobacterial species. Conclusions: Together, these findings provide valuable insights, which could accelerate the development of intervention strategies for this critical pathway pivotal in the progression of M. tuberculosis infection.