Conformational dynamics in human neuroglobin: effect of His64, Val68, and Cys120 on ligand migration.

Neuroglobin belongs to the family of hexacoordinate hemoglobins and has been implicated in the protection of neuronal tissue under hypoxic and ischemic conditions. Here we present transient absorption and photoacoustic calorimetry studies of CO photodissociation and bimolecular rebinding to neuroglobin focusing on the ligand migration process and the role of distal pocket residues (His64 and Val68) and two Cys residues (Cys55 and Cys120). Our results indicate that His64 has a minor impact on the migration of CO between the distal heme pocket and protein exterior, whereas the Val68 side chain regulates the transition of the photodissociated ligand between the distal pocket and internal hydrophobic cavities, which is evident from the increased geminate quantum yield in this mutated protein (Φ(gem) = 0.32 for WT and His64Gln, and Φ(gem) = 0.85 for Val68Phe). The interface between helix G and the A-B loop provides an escape pathway for the photodissociated ligand, which is evident from a decrease in the reaction enthalpy for the transition between the CO-bound hNgb and five-coordinate hNgb in the Cys120Ser mutant (ΔH = -3 ± 4 kcal mol(-1)) compared to that of the WT protein (ΔH = 20 ± 4 kcal mol(-1)). The extensive electrostatic/hydrogen binding network that includes heme propionate groups, Lys67, His64, and Tyr44 not only restricts the heme binding but also modulates the energetics of binding of CO to the five-coordinate hNgb as substitution of His64 with Gln leads to an endothermic association of CO with the five-coordinate hNgb (ΔH = 6 ± 3 kcal mol(-1)).