Oxygen entry through multiple pathways in T-state human hemoglobin

The heme oxygen (O₂) binding site of human hemoglobin (HbA) is buried in the interior of the protein, and there is a debate over the O ₂ entry pathways from solvent to the binding site. As a first step to understand HbA O₂ binding process at the atomic level, we detected all significant multiple O₂ entry pathways from solvent to the binding site in the α and β subunits of the T-state tetramer HbA by utilizing ensemble molecular dynamics (MD) simulation. By executing 128 independent 8 ns MD trajectories in O₂-rich aqueous solvent, we simulated the O₂ entry processes and obtained 141 and 425 O ₂ entry events in the α and β subunits of HbA, respectively. We developed the intrinsic pathway identification by clustering method to achieve a persuasive visualization of the multiple entry pathways including both the shapes and relative importance of each pathway. The rate constants of O₂ entry estimated from the MD simulations correspond to the experimentally observed values, suggesting that O₂ ligands enter the binding site through multiple pathways. The obtained multiple pathway map can be utilized for future detailed analysis of HbA O₂ binding process.