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

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 t( 1/4 ) 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.