Resolving the Transition States of Human Hemoglobin Assembly Through a Combination of Spectroscopic Studies and All-Atom Molecular Dynamics Simulations

The assembly of hetero-tetrameric HbA is a multi-faceted pathway emphasized by both quaternary structure formations via the interacting alpha and beta subunits of HbA as well as the oxygen coordinating heme (Fe(II)-protoporphyrin IX) insertion within the heme cavity of each subunit. Folding transition states at different stages of this pathway could act as precursors for misassembly of HbA, leading to hemoglobinopathies and disruption of oxygen transport within the cardiovascular system. Simultaneous circular dichroism and visible absorbance measurements of guanidine hydrochloride induced unfolding of HbA showed that the initial formation of the alpha-beta dimer interface occurs via a molten globule heterodimer state with ∼30% helical content. Reversible hemin (Fe(III)-protoporphyrin IX) binding to the melted heme pockets in this transition state results in a six coordinate, low spin iron state known as a hemichrome, which stabilizes the hetero-dimer interface. Atomic level modeling of hemin disassociation from native HbA using the Amber 2018 molecular dynamics (MD) package showed that these hemichrome transition states can also occur in both the folded alpha and beta subunits at 37 °C via hexacoordination of the iron by internal histidines within the heme cavity. These atomic level simulations enable us to characterize the transitionary nature of bond breaking and formation with metal cofactors that is beyond the resolution limits of solution spectroscopy and X-ray crystallography. Modeling studies involving thermodynamics integration are also currently being undertaken to measure free energy change for HbA tetramer interface formation, which markedly enhances heme affinity of HbA.