Atomistic Simulations of Heme Dissociation Pathways in Human Methemoglobins Reveal Hidden Intermediates.

Heme dissociations disrupt function and structural integrity of human hemoglobin and trigger various cardiovascular complications. These events become significant in methemoglobins that have undergone autoxidation of ferrous into ferric heme. We have structurally characterized the heme disassociation pathways for adult tetrameric methemoglobins using all-atom molecular dynamics simulations. These reveal that bis-histidine hemichromes, characterized here by the coordination of heme iron to both the F8 (proximal) and E7 (distal) histidines, are seen as intermediates following dissociation of the water molecule distally bound to each heme iron. Later, the breaking of coordination between heme iron and proximal histidine disrupts the F helix and pushes it away from the heme cavity, enabling both bulk solvent penetration and disruption of tetramer interface interactions. The interactions inhibiting heme dissociation were then seen to be (i) either a direct or a water-molecule-mediated interaction between distal histidine and heme iron and (ii) stacking between heme and the αCE1/βCD1 phenylalanine residue. These interactions are less important in the β than in α subunits due to a more flexible β subunit CE loop region. The absence of a distal histidine interaction in the H(E7)L mutant and increased heme cavity volume in the V(E11)A mutant both promoted heme escape from the protein interior. Adult and fetal hemoglobins were seen to share a general heme disassociation pathway and intermediates due to the conservation of key heme pocket residues. The intermediates seen here are analyzed in light of experimental studies of heme dissociation and pathways of certain hemoglobinopathies.