Our overall goal is to prepare and characterize metal-free cytochrome c, so-called porphyrin cytochrome c (H₂cyt), and its various metal-substituted derivatives to better understand the role of the metal ion on structural stability in proteins. The removal of the native iron in cytochrome c and substitution by different metal ions affects the local coordination environment, overall structural stability, and biological function. Our specific aim is to optimize the conditions for incorporation of a cobalt(II) or manganese(II) ion into H₂cyt. We vary the temperature and concentrations of the metal ion and denaturant (guanidine hydrochloride or urea). Compared to H₂cyt, metal-substituted cytochromes have significantly different absorbance spectra, so we monitor over time the change in absorbance upon addition of the metal ion. In the presence of increased temperature and high denaturant concentration, we observed complete incorporation of both cobalt(II) and manganese(II). The rate constants for incorporation of cobalt(II) into H₂cyt at 5.0 M urea increased dramatically above 55°C. However, the rate constant for incorporation of cobalt(II) into H₂cyt at 8.0 M urea was maximal at only 40°C; increased temperature decreased the rate of incorporation. Overall, the rate of incorporation of manganese(II) into H₂cyt is slower than the rate of incorporation of cobalt(II).