A differential scanning calorimetric study of the thermal unfolding of apo‐ and holo‐cytochrome b₅₆₂

Abstract

Cytochrome b562 is a four-helix-bundle protein containing a non-covalently bound b-type heme prosthetic group. In the absence of heme, cytochrome b562 remains highly structured under native conditions. Here we report thermodynamic data for the thermal denaturation of the holo-and apoproteins as determined by differential scanning calorimetry. Thermal denaturation of holocytochrome b562 is a highly reversible process, and unexpectedly does not involve dissociation of the heme prosthetic group. Thermal denaturation of the corresponding apoprotein, with the heme group chemically removed, remains a cooperative, reversible process. Apocytochrome b562 is substantially destabilized relative to the holoprotein: the tl12 is more than ten degrees lower, and enthalpy and heat capacity changes are about one-half of the holoprotein values. However, the energetic parameters of apocytochrome b562 denaturation are within the range of observed values for small proteins.

Effects of Heme on the Structure of the Denatured State and Folding Kinetics of Cytochrome b562

“…Finally, as a result of calorimetric studies, it has been suggested that heme is still associated with the thermally denatured state of ferric cytochrome b 562 (K d 6 mM). 39,40 All the thermodynamic evidence suggests that the apparently denatured state of the reduced cytochrome is clearly more collapsed, as we have revealed by our NMR analysis.…”

Multiple Step Assembly Of The Transmembrane Cytochrome b6

“…7 Transmembrane b-type cytochromes contain noncovalently bound heme cofactors, and the proteins are involved in charge transfer processes, such as photosynthetic and respiratory electron transfer chains. While in the recent years folding, assembly, and stability of soluble b-type cytochromes, [9][10][11][12][13][14] or of synthetic soluble b-type cytochrome models [15][16][17][18][19][20] have been studied to some extent, not much is known about the assembly and stability of membrane-integrated b-type cytochromes. 8 For the transmembrane cytochrome b 559 ′ it has been clearly shown that the apo-protein assembles completely independent of the cofactor, and in a subsequent stage a single heme molecule binds noncovalently to a preformed transmembrane helix dimer.…”

Heme binding properties of heterologously expressed spinach cytochrome b6: Implications for transmembrane b‐type cytochrome formation

“…In several studies soluble representatives have been used to analyze and describe folding, assembly, and stability of a b-type cytochrome (e.g. [1][2][3][4][5][6]). Furthermore, by the use of designed heme proteins many aspects of the structure and function of b-type cytochromes have been addressed and the structural basis for cytochrome assembly and function of the heme cofactors has been analyzed to some extend (for recent reviews see [7,8]).…”