Acid denaturation studies on a cobalt(3) protoporphyrin-globin complex.

A cobalt (III)-protoporphyrin IX complex of horse globin (cobaltiglobin) has been prepared and its stability toward acid denaturation determined. Unlike iron(II1) protoporphyrin, which combines with. globin to give ferrihemoglobin, cobalt(II1) protoporphyrin combines with globin to give a complex which experimentally resembles an internal ferrihemichrome in which the heme is attached to the protein by two metal protein bonds. The evidence for this conclusion is that, unlike ferrihemoglobin, cobaltiglobin does not combine with cyanate, thiocyanate, fluoride, or azide. It does combine with cyanide, although slowly. It has no ionizable water coordinated to the metal. No significant change in spectrum occurs in 12 hours at pH values as low as pH 3.65, but pa-stat experiments reveal that two basic groups per dimer are slowly unmasked at pH values as high as pH 5. Unlike the denaturation of all other hemoglobin complexes studied, the pH profile of cobaltiglobin shows that the denaturation rate is only slightly dependent on pH (proportional to about [H+]0.37). The effect of temperature is small also. Unlike the heme in ferrihemoglobin, the cobalt(II1) porphyrin remains attached to the polypeptide chain when the latter is unfolded. In this respect it resembles CO-hemoglobin. The observed data are compatible with the internal hemichrome formulation and indicate that substitution of the metal can have a profound influence on the structure of the protein.