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, absorption changes associated with altered ligation in the alkaline isomers were observed. In addition, the intensity of the 695-nm absorption band, which is associated with the heme-crevice stability, decreased with increasing temperature and exhibited biphasic temperature dependence, with transition temperatures (Tc) at 35 degrees C in tuna c, 55 degrees C in horse c, and 58 C in bovine c. Since the heme crevice plays a key role in the thermal stabilities of cytochromes c, their susceptibility to proteolytic attack was examined as a function of temperature. Proteolytic digestion, which requires local conformational instability, revealed that the local stabilities of the cytochromes follow the order: bovine > horse >> tuna, and increased digestion occurred at temperatures close to the 695-nm Tc for each protein. This is consistent with the actual substitution of the Met80 ligand above the 695-nm Tc, which is reflected in the thermodynamic parameters for the two phases. Also, tuna c, unlike horse and bovine c, exhibits different 695-nm (35 degrees C) and Soret (approximately 46 degrees C) Tc values, but its local stability is controlled by the transition detected at 695 nm. The combined spectroscopic and proteolysis results clearly indicate that the flexibility of the Met80 loop determines the local stability of cytochromes c.
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Department of Chemistry and Biochemistry, Concordia University, West Montreal, Quebec, Canada.
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D020816:Amino Acid Motifs; D000818:Animals; D002417:Cattle; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism; D006736:Horses; D006863:Hydrogen-Ion Concentration; D009206:Myocardium / Q000737:chemistry; D011487:Protein Conformation; D013696:Temperature; D013816:Thermodynamics; D014413:Tuna
D003574:Cytochrome c Group