Bacterial chemotaxis, the directed movement of bacteria in a chemical environment, represents one of the best biochemically and structurally characterized signal transduction pathways. The histidine kinase CheA is a central player in this two component regulatory system. Its active site is spread across two domains: the histidine phosphotransfer domain (P1) and the kinase domain (P4). Our efforts focus on elucidating the mechanistic contribution of P1 residues to the autophosphorylation reaction. An atomic resolution structure (0.98 Å) of the Thermotoga maritima CheA histidine phosphotransfer domain was obtained, affording a unique opportunity to view the environment surrounding His45, the phosphoaccepting histidine, in detail. His45, participates in a hydrogen bonding network including three other residues: Glu67, Lys48, and His64, which are conserved in CheA. Employing a combination of site-directed mutagenesis studies, protein crystallography, and 2D heteronuclear NMR techniques, we explored the functional roles of these residues involved in the largely conserved hydrogen bonding network. Our experiments revealed that the P1 domain provides the nucleophile for phosphate transfer (His45) and the activating glutamate (Glu67) completing a catalytic center observed in the GHL family of ATPases. Glu67 tunes the reactivity of His45 through a hydrogen bond. This interaction activates His45 to the normally unfavored N?1H tautomeric state. As a result, His45 possesses an altered pKa. Upon mutation of Glu67 to a Gln, the chemical properties of His45 change. When existing in the predominantly Nε2H tautomeric state, its pKa is similar to that of a solvent exposed histidine and its phosphorylation is dramatically reduced in vitro and in vivo. Hence, the phosphoaccepting histidine must exist in the normally unfavored N?1H tautomeric state in order for CheA autophosphorylation to occur. The other two residues, Lys48 and His64, do not affect the reactivity of His45. Instead they contribute towards the structural integrity of the P1 active site. The results obtained in this thesis provide a solid structural and biochemical basis for further understanding the CheA phosphotransfer mechanism and may provide critical insight for the development of novel antibiotic agents.