Recent discoveries of a number of hypertrophic cardiomyopathy related mutations in the C-terminal domain of cardiac troponin C suggest that sites III and IV might play a more important role than just anchoring troponin C into the troponin complex. We investigated the effects of hypertrophic cardiomyopathy related mutation D145E in human cardiac troponin C on calcium binding and exchange with the C-terminal domain sites III and IV. The calcium titration data indicated that the D145E substitution in the +z position of the calcium binding site IV dramatically decreased calcium binding affinity of that site (∼1, 856-fold), and virtually eliminated magnesium binding to that site. Furthermore, the D145E substitution significantly decreased the calcium affinity of site III (∼1.4-fold), correlating with ∼1.6-fold faster rate of calcium dissociation from site III. Stopped-flow studies utilizing fluorescent calcium chelator Quin-2 demonstrated that the D145E mutation reduced the stoichiometry of moles of calcium per mole of the C-terminal domain by ∼2-fold, both in the absence and presence of cardiac troponin I peptide (residues 34-71). Thus, binding of troponin I peptide to the C-terminal domain of D145E troponin C was not able to restore normal calcium binding to site IV. These results indicate the conservative D145E substitution has detrimental effects on calcium and magnesium binding to site IV of troponin C.