Hemopexin alters conformation upon binding
heme as shown by circular dichroism (CD), but
hemopexin binds the
heme analog, iron-meso-tetra-(4-sulfonatophenyl)-porphine (FeTPPS), without undergoing concomitant changes in its CD spectrum. Moreover, FeTPPS, unlike
heme, does not increase the compactness of the
heme-binding domain (I) of
hemopexin shown by an increased sedimentation rate in
sucrose gradients. On the other hand, like
heme, FeTPPS forms a bishistidyl coordination complex with
hemopexin and upon binding protects
hemopexin from cleavage by
plasmin. Competitive inhibition and saturation studies demonstrate that FeTPPS-
hemopexin binds to the
hemopexin receptor on mouse
hepatoma cells but with a lower affinity (Kd 125 nM) more characteristic of apo-
hemopexin than
heme-
hemopexin (Kd 65 nM). This provides evidence that conformational changes produced in
hemopexin upon binding
heme, but not upon binding FeTPPS, are important for increasing the affinity of
hemopexin for its receptor. The amount of cell-associated radiolabel from 55FeTPPS-hemopexin increases linearly for up to 90 min but at a rate only about a third of that of the
mesoheme-complex. As expected from the recycling of
hemopexin, more
iron-
tetrapyrrole than
protein is associated with the Hepa cells, but the ratio of 55Fe-ligand to 125I-hemopexin is only 2:1 for FeTPPS-
hemopexin compared to 4:1 for
mesoheme complexes. [55Fe]
Mesoheme was associated at 5 min with lower density fractions containing plasma membranes and at 30 min with fractions containing higher density intracellular compartments. In contrast, 55FeTPPS was found associated with plasma membrane fractions at both times and was not transported into the cell. Although FeTPPS-
hemopexin binds to the receptor, subsequent events of
heme transport are impaired. The results indicate that upon binding
heme at least three types of conformational changes occur in
hemopexin which have important roles in receptor recognition and that the nature of the
ligand influences subsequent
heme transport.