FTIR-monitored thermal titration reveals different mechanisms for the alkaline isomerization of tuna compared to horse and bovine cytochromes c.

Fourier transform infrared (FTIR) spectroscopy is used to compare the thermally induced conformational changes in horse, bovine and tuna ferricytochromes c in 50 mM phosphate/0.2 M KCl. Thermal titration in D2O at pD 7.0 of the amide II intensity of the buried peptide NH protons reveals tertiary structural transitions at 54 degrees C in horse and at 57 degrees C in bovine c. These transitions, which occur well before loss of secondary structure, are associated with the alkaline isomerization involving Met80 heme-ligand exchange. In tuna c, the amide-II-monitored alkaline isomerization occurs at 35 degrees C, followed by a second amide II transition at 50 degrees C revealing a hitherto unreported conformational change in this cytochrome. Amide II transitions at 50 degrees C (tuna) and 54 degrees C (horse) are also observed during the thermal titration of the CN(-)-ligated cytochromes (where CN- displaces the Met80 ligand), but a well-defined 35 degrees C amide II transition is absent from the titration curve of the CN- adduct of tuna c. The different mechanisms suggested by the FTIR data for the alkaline isomerization of tuna and the mammalian cytochromes c are discussed. After the alkaline isomerization, loss of secondary structure and protein aggregation occur within a 5 degrees C range with Tm values at 74 degrees C (bovine c), 70 degrees C (horse c) and 65 degrees C (tuna c), as monitored by changes in the amide I' bands. The FTIR spectra were also used to compare the secondary structures of the ferricytochromes c at 25 degrees C. Curve fitting of the amide I (H2O) and amide I' (D2O) bands reveals essentially identical secondary structure in horse and bovine c, whereas splitting of the alpha-helical absorption of tuna c indicates the presence of less-stable helical structures. CN- adduct formation results in no FTIR-detectable changes in the secondary structures of either tuna or horse c, indicating that Met80 ligation does not influence the secondary structural elements in these cytochromes. The data provided here demonstrate for the first time that the selective thermal titration of the amide II intensity of buried peptide NH protons in D2O is a powerful tool in protein conformational analysis.