Calorimetric studies of the thermal denaturation of cytochrome c peroxidase

Abstract

Two endotherms are observed by differential scanning calorimetry during the thermal denaturation of cytochrome c peroxidase at pH 7.0. The transition midpoint temperatures (tm) were 43.9 +/- 1.4 and 63.3 +/- 1.6 degrees C, independent of concentration. The two endotherms were observed at all pH values between 4 and 8, with the transition temperatures varying with pH. Precipitation was observed between pH 4 and 6, and only qualitative data are presented for this region. The thermal unfolding of cytochrome c peroxidase was sensitive to the presence and ligation state of the heme. Only a single endotherm was observed for the unfolding of the apoprotein, and this transition was similar to the high-temperature transition in the holoenzyme. Addition of KCN to the holoenzyme increases the midpoint of the high-temperature transition whereas the low-temperature transition was increased upon addition of KF. Binding of the natural substrate ferricytochrome c to the enzyme increases the low-temperature transition by 4.8 +/- 1.3 degrees C but has no effect on the high-temperature transition at pH 7. The presence of cytochrome c peroxidase decreases the stability of cytochrome c, and both proteins appear to unfold simultaneously. The results are discussed in terms of the two domains evident in the X-ray crystallographic structure of cytochrome c peroxidase.

Structural and thermodynamic consequences of b heme binding for monomeric apoglobins and other apoproteins

“…The thermal denaturation of the holoprotein proceeds with two distinct transitions; the apoprotein exhibits only one, matching the higher temperature transition of the holoprotein. The molecular origin of these transitions, however, does not appear to correspond to the domain structure (Kresheck and Erman, 1988). ApoCCP can form crystals (Yonetani, 1967), and the structure of the iron-free protein has been solved (Su et al, 1994;Bhaskar and Poulos, 2005).…”

Designing Protein Denaturants: Synthetic Agents Induce Cytochromec Unfolding at Low Concentrations and Stoichiometries

“…Consequently, silver is expected to adopt subvalent valence states in solid materials. [3] Only a few examples such as, Ag 2 F, [4] Ag 3 O, [5] Ag 5 SiO 4 , [6,7] Ag 5 GeO 4 , [6,8] and Ag 5 Pb 2 O 6 [9] have been prepared to date. The properties range from metallic behavior in Ag 2 F, Ag 3 O, and Ag 5 Pb 2 O 6 to semiconducting in Ag 5 SiO 4 and Ag 5 GeO 4 .…”

Effect of polyglutamate on the thermal stability of ferricytochrome c

“…From microcalorimetric measurements it is evident that the redox partners of cytochrome c, such as cytochrome c oxidase [11], cytochrome c peroxidase [12] as well as cytochrome c~ [13] decrease the melting point of ferricytochrome c. Unfortunately, these complexes lose the reversibility and it is not possible to exactly treat the thermodynamic data. Surprisingly, heparin, a linear hydrophilic polyanion, has a very similar effect as the redox partners on the thermal stability of ferricytochrome c [14].…”