LAUSR

Deriving the pathway for human hemoglobin assembly is important both for developing treatments for hemoglobinopathies and for designing robust acellular hemoglobin oxygen carriers. The adult human hemoglobin (HbA) comprises two globin heterodimers, (α1β1)(α2β2), with each subunit containing a heme group for coordination of oxygen and having a tertiary structure very similar to the monomeric paralog, myoglobin.In order to determine the complete folding mechanism, we have measured and analyzed guanidinium-induced unfolding curves for both apo- and holo-hemoglobin. This approach expands on previous work done in our lab with myoglobin and has allowed us to deconvolute the globin unfolding pathway and the heme-binding effects for the various folding states. When heme is extracted from human Hb, the resulting apohemoglobin dissociates to an α1β1 heterodimer. Unfolding occurs in two major phases. The first involves melting of the heme pockets to generate a dimeric molten globule, followed by dissociation into almost completely unfolded monomers. Structural identification of these phases was achieved by comparing the unfolding curves for native HbA, recombinant HbF, a N-to-C termini linked α Hb variant, and a series of Hb mutants with large, hydrophobic residues replacing the distal histidine and valine in the α and β heme pockets.Previously, we showed that monomeric apoglobin stability, and not hemin affinity, is the determining factor for myoglobin expression. In the case of hemoglobin, the individual monomers are unstable, but associate strongly to form a stable molten globule dimeric state, which binds hemin and facilitates the overall assembly of the hemoglobin.Supported by the NIH Grant HL110900 and by Grant C-0612 from Robert A. Welch Foundation.