Metacaspase proteins have been identified in fungi, plants, and protozoa and are thought to play roles in programmed cell death pathways, pathogenicity, and stress responses. This project studies a Type I metacaspase from the fungus Schizophyllum commune (ScMCA-Ia). These metacaspases, like many others, require calcium for activation. A well-characterized ScMCA-Ia site binds calcium with a binding constant in the micromolar range, but ScMCA-Ia only reaches maximal activity once calcium concentration is in the millimolar range. This leads to a hypothesis that there are two calcium binding sites in ScMCA-Ia. Circular dichroism (CD) spectroscopy gives insight into the interaction between ions and protein. Secondary structural elements of proteins give strong signals at unique wavelengths due to their interactions with circularly polarized light. We observe a change in signal at 222nm, the characteristic wavelength of alpha helical interactions with the light, as we increase calcium or other cation concentration in the protein sample, suggesting a conformational change due to calcium/cation binding. This data can be analyzed and fit to determine binding constants. In addition to calcium, other divalent cations have also been studied. While these ions may not activate the metacaspase, their binding abilities can help to better understand the metacaspase interactions with cations. Preliminary results show that calcium, manganese, nickel, and cobalt bind to two sites in the structure, while magnesium does not bind. Additionally, kinetic fluorescence assays provide information about the maximal activity and enzyme efficiency as calcium concentration is varied. Determining the value of kcat at a variety of concentrations of calcium also allows us to determine binding constants. Further work will develop an improved protocol for analysis of the CD spectroscopy data that can be fit with a nonlinear regression to determine binding constants for each of the divalent cations studied with ScMCA-Ia. These results will be compared to the values of the binding constants obtained with the kinetic fluorescence assays.