The properties of wild-type, V68T, and H97D sperm whale myoglobins were compared to determine the relative importance of heme affinity and globin stability on the resistance of the holoprotein to denaturation. The V68T mutation decreases apoglobin stability by placing a polar side chain in the interior heme pocket. However, this substitution increases hemin affinity by formation of a strong hydrogen bond between coordinated water and the Thr(68)(E11) side chain. The H97D substitution disrupts favorable contacts with Ser(92)(F7) and the heme-7-propionate and causes a large increase in the rate of hemin dissociation. The Asp replacement has little affect on apoglobin stability because His(97)(FG3) is a surface residue. The aquomet, cyanomet, deoxyferrous, and apoglobin forms of each mutant and wild-type myoglobin were unfolded by titration with guanidinium chloride. Even though holomyoglobin denaturation involves the dissociation of heme and should be dependent on protein concentration, nonspecific heme binding to unfolded states makes the overall process appear to be a simple, unimolecular unfolding transition. The equilibrium constants for the denaturation of the holomyoglobin mutants correlate almost exclusively with heme affinity and not with the stability of the globin portion of the molecule. The strong correlation with heme affinity explains quantitatively why the stability of myoglobin is enhanced similar to 60-fold by reduction of iron to the ferrous deoxy state and by another similar to 100-fold with CO coordination. Parameters measured for GdmCl-, urea-, acid-, and heat-induced denaturation of holomyoglobins and hemoglobins reflect heme affinity and not the folding properties of the corresponding apoproteins. This conclusion suggests that (1) many previous studies of the denaturation of intact heme proteins need to be reevaluated in terms of heme affinity and (2) measurements with apoproteins are required for unambiguous determinations of the stability of globin structures.
