The interplay between molten globule intermediates and heme disassociation characterizes human hemoglobin disassembly

Background: Hemoglobin functions as an oxygen transport protein via its hetero-tetrameric structure, with each subunit containing an iron-cofactor heme. We have developed a global disassembly model for human hemoglobin, linking hemin (ferric heme) disassociation and apo (heme-free) subunits unfolding pathways. Results: Model evaluation was done using circular dichroism and visible absorbance spectroscopy measurements of guanidine hydrochloride-induced holo (heme-bound) hemoglobin disassembly and our previous measurements of apohemoglobin unfolding. The populations of holo intermediates and equilibrium disassembly parameters were determined quantitatively for adult and fetal hemoglobins. Our results show that the key stages for disassembly into unfolded monomers are characterized by hemichrome intermediates with molten globule characteristics. Hemichromes, which occur when both hemin iron axial sites coordinate amino acids, are not energetically favored in native human hemoglobins. However, we have determined that these hexacoordinate iron complexes are important for preventing hemin disassociation from partially unfolded species during early disassembly. Both our model evaluation and independent small angle X-ray scattering measurements demonstrate that heme disassociation during early disassembly leads to loss of the tetrameric structural integrity. Our model predicts that dimeric and monomeric hemichrome intermediates mediate the disassembly pathway inside red cells where the hemoglobin concentration is very high. This prediction explains why in the red cells of patients with unstable hemoglobinopathies misassembled hemoglobins often get trapped as hemichromes, accumulate into insoluble Heinz bodies, become deposited at the cell membranes, and often lead to hemolysis. Alternatively, when acellular hemoglobin is diluted into blood plasma after red cell lysis, the disassembly pathway is dominated by early hemin disassociation from the unfolding intermediates. This leads to the generation of higher fractions of apo subunits and free hemin, which is known to damage to the integrity of blood vessel walls. Conclusions: Our model demonstrates the existence of alternate sets of disassembly/assembly pathways for human hemoglobin, illuminates the pathophysiology of some hemoglobinopathies and other disease states associated with unstable globins and red cell lysis, and provides insights into the factors governing hemoglobin assembly during erythropoiesis.