Energetics of heme binding to native and denatured states of cytochrome b562

Cytochrome b(562) is a four-helix bundle protein containing a noncovalently bound b-type heme prosthetic group. For the first time, energetics of heme binding to an apocytochrome were measured by isothermal titration calorimetry. The heme is tightly bound to native apocytochrome b(562), With a dissociation constant (K-d) Of approximately 9 nM (Delta G degrees = 11 kcal mol(-1)) at 25 degrees C. Unexpectedly, the thermally denatured apoprotein is also capable of specifically binding heme with modest affinity (K-d = 3 mu M, Delta G degrees = 7.6 kcal mol(-1)). This interaction results in the dependence of holocytochrome b(562) stability on protein concentration in the submicromolar range.