Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, infects nearly 10 million people each year and is responsible for the death of 1.5 million people annually. The bacteria encodes several metal transport systems that are critical for its survival in phagosomes where the metal concentrations are always changing. The bacteria have two metalloregulators that are associated with Ni(II) and Co(II) export, NmtR and KmtR. Nothing is known about why KmtR is responsive to Ni(II) and Co(II) binding and if KmtR is capable of binding to other first-row transition metals.The fact that M. tuberculosis has two regulators that are associated with the expression of two different exporters for Ni(II) and Co(II) suggests that maintaining the intracellular Ni(II) and Co(II) concentrations is critical to the bacteria. Metal binding studies conducted using Isothermal Titration Calorimetry (ITC) have determined that KmtR binds Ni(II) and Co(II) with nanomolar affinity. The goal of our research is to identify the residues that are responsible for binding the cognate metals, Ni(II) and Co(II), as well as the noncognate metal, Zn(II), to KmtR. Mutagenesis studies coupled with metal binding experiments are being utilized to determine how KmtR binds these metals. The H88Q, E101Q, H102Q, and H111Q mutants, among others, have been made, expressed, and purified in our lab. Data obtained from ITC determined that like the WT protein, the E101Q and H102Q mutant proteins bind Co(II) with nanomolar affinity and the H88Q and H111Q mutant KmtR proteins bind Co(II) with micromolar affinity.