Probing local thermal stabilities of bovine, horse, and tuna ferricytochromes c at pH 7

Correlation between the flexibility of the Met80 loop (residues 75-86) and the local stabilities of native ferricytochromes c from horse, bovine, and tuna was examined. By monitoring the heme bands versus temperature, absorpt ion changes associated with altered ligation in the alkaline isomers were o bserved. In addition, the intensity of the 695-nm absorption band, which is associated with the heme-crevice stability, decreased with increasing temp erature and exhibited biphasic temperature dependence, with transition temp eratures (T-c) at 35 degrees C in tuna c, 55 degrees C in horse c, and 58 d egrees C in bovine c. Since the heme crevice plays a key role in the therma l stabilities of cytochromes c, their susceptibility to proteolytic attack was examined as a function of temperature. Proteolytic digestion, which req uires local conformational instability, revealed that the local stabilities of the cytochromes follow the order: bovine>horse>> tuna, and increased di gestion occurred at temperatures close to the 695-nm T-c for each protein. This is consistent with the actual substitution of the Met80 ligand above t he 695-nm T-c, which is reflected in the thermodynamic parameters for the t wo phases. Also, tuna c, unlike horse and bovine c, exhibits different 695- nm (35 degrees C) and Soret (similar to 46 degrees C) T-c values, but its l ocal stability is controlled by the transition detected at 695 nm. The comb ined spectroscopic and proteolysis results clearly indicate that the flexib ility of the Met80 loop determines the local stability of cytochromes c.