Molten Globules and Metallocofactor Disassociation Steer Human Hemoglobin Disassembly

We depend on the protein hemoglobin to transport oxygen from our lungs to our cells. Functional hemoglobin is a heterotetramer with each subunit encapsulating a metallocofactor heme within a cavity. Hemoglobinopathies resulting in cardiovascular complications could manifest via hemoglobin misassembly or instability events. These events can be triggered by accumulation of unstable disassembly intermediates or heme disassociation, facilitated following ferrous heme auto-oxidation into ferric heme (hemin). We used a combination of spectroscopy and molecular dynamics (MD) simulations approaches to resolve structural mechanisms of human hemoglobin disassembly, uncover hidden disassembly intermediates, and identify potential hemoglobinopathy therapeutic targets. Measurements of guanidine hydrochloride triggered reversible disassembly of hemoglobin were done of protein secondary structure loss through circular dichroism; and hemin environment changes through visible absorbance. Using these spectral measurements, we then developed a quantitative hemoglobin disassembly model that predicts the disassembly pathway would differ markedly between erythrocytes and acellular blood plasma environments. In hemoglobin packed erythrocytes, molten globule intermediates with hemichrome characteristics will dominate the pathway. Hemichromes have been implicated in hemoglobinopathies and are characterized by amino acid coordination at each of the hemin iron axial site. With cell lysis, hemoglobin dilution into plasma will promote hemin disassociation. Atomic level MD simulations were then performed to probe hemin disassociation pathways in folded hemoglobins. The simulations revealed that local protein backbone arrangements around the heme cavities sometimes lead to a bis-histidine hemichrome as an on-pathway intermediate. This intermediate has not been experimentally resolved and was seen to dampen hemin disassociation. We also discovered new electrostatics and pi-stacking interactions between hemin and certain residues lining the heme cavity. Thermodynamics integration method of MD simulations is independently being used to evaluate inter-subunit interactions and energetics related to destabilization of hemoglobin tetramer interfaces resulting from clinically relevant mutations.