Staphylococcus aureus is a medically important Gram-positive bacterial pathogen that actively procures heme from human hemoglobin (Hb) using the iron-regulated surface determinant (Isd) system. Research described in this dissertation investigated how the Isd system uses the IsdH receptor protein to capture Hb and extract its hemin (the oxidized form of heme). To rapidly extract Hb’s hemin, IsdH employs a conserved tri-domain unit that contains two NEAr iron Transporter (NEAT) domains that are connected by a helical linker domain. The work described in chapter 2 used UV-Vis spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and electrospray ionization mass spectrometry (ESI-MS) methods to define the importance of the conserved linker domain in hemin extraction and revealed that this domain enables the NEAT domains within the tri-domain unit to work together to synergistically extract hemin. Chapter 3 of this thesis describes the structure and dynamics of the tri-domain unit (IsdHN2N3). The solution structure of the apo-receptor was defined using small angle X-ray scattering, and advanced NMR methods such as paramagnetic relaxation enhancement (PRE), residual dipolar coupling (RDC), and selective methyl labeling approaches. The structure and inter-domain dynamics of IsdHN2N3 in the absence of Hb were further defined using ensemble modeling calculations. The results of these studies illustrated that the receptor adaptively recognizes Hb using a combination of conformational selection and induced fit mechanisms, and suggests that the linker domain may facilitate hemin transfer by destabilizing the iron-coordinating F-helix in Hb. Chapter 4 describes studies that investigated the kinetic and thermodynamic basis of hemin transfer using stopped-flow UV-Vis spectroscopy, analytical ultracentrifugation sedimentation equilibrium, and isothermal titration methods. The results of this work provide insight into the kinetic and thermodynamic determinants that facilitate receptor-mediated hemin release from Hb. Lastly, Chapter 5 describes the methods that were used to site-specifically label proteins with nitroxide spin-label probes and the subsequent derivation of paramagnetic NMR distance restraints. Altogether, the results of the work described in this dissertation have advanced our knowledge of Hb recognition and hemin acquisition by the pathogen S. aureus.