Quaternary-Linked Changes in Structure and Dynamics That Modulate O₂ Migration within Hemoglobin’s Gas Diffusion Tunnels

Abstract

Atomistic molecular dynamics simulations of diffusion of O2 from the hemes to the external solvent in the α- and β-subunits of the human hemoglobin (HbA) tetramer reveal transient gas tunnels that are not seen in crystal structures. We find here that the tunnel topology, which encompasses the reported experimental Xe binding cavities, is identical in HbA's T, R, and R2 quaternary states. However, the O2 population in the cavities and the preferred O2 escape portals vary significantly with quaternary structure. For example, most O2 molecules escape from the T β-subunit via the cavity at the center of the tetramer, but direct exit from the distal heme pocket dominates in the R2 β-subunit. To understand what triggers the quaternary-linked redistribution of O2 within its tunnels, we examined how the simulated tertiary structure and dynamics of each subunit differs among T, R, and R2 and report that minor adjustments in α-chain dynamics and β-heme position modulate O2 distribution and escape in HbA. Coupled to the β-heme position, residue βF71 undergoes quaternary-linked conformations that strongly regulate O2 migration between the β-subunit and HbA's central cavity. Remarkably, the distal histidine (HisE7) remains in a closed conformation near the α- and β-hemes in all states, but this does not prevent an average of 23, 31, and 46% of O2 escapes from the distal heme pockets of T, R, and R2, respectively, via several distal portals, with the balance of escapes occurring via the interior tunnels. Furthermore, preventing or restricting the access of O2 to selected cavities by mutating HisE7 and other heme pocket residues to tryptophan reveals how O2 migration adjusts to the bulky indole ring and sheds light on the experimental ligand binding kinetics of these variants. Overall, our simulations underscore the high gas porosity of HbA in its T, R, and R2 quaternary states and provide new mechanistic insights into why undergoing transitions among these states likely ensures effective O2 delivery by this tetrameric protein.

O2 and Water Migration Pathways between the Solvent and Heme Pockets of Hemoglobin with Open and Closed Conformations of the Distal HisE7

“…9,12 In the six HbA models examined here, R c , HbA were chosen as the initial structures for the T (2DXM) 13 and R (2DN3) 14 models, respectively, and the T c and R c results were obtained with the distal HisE7 in the neutral N ε2 -H tautomer, 12 which remained in the closed conformation found in the crystal structures for 99% of the MD simulation time ( Figure 1a,d,g; S1). T o and R o were modeled by HisE7 protonation in each subunit.…”

“…The initial T -and R -structures of the HbA(α,βHisE7Gly) variant were built from wild-type HbA by removing the HisE7 side chain ( Figure 5 c,f,i). The CO and distal water molecules were removed to allow placement of the O 2 copies in the distal heme sites, and after structure equilibration, all-atom MD simulations of O 2 diffusion were performed in explicit solvent at 310 K. Since the ligands do not migrate between the subunits, 9,12 15 TLES O 2 copies were placed in the α-or β-distal site of HbA. Thirty-two replicate simulations of 2-ns duration provide 480 O 2 trajectories that separately map gas migration within each subunit for the six HbA models defined here (Table S1).…”

“…4,5,8 We observe that the neutral form of HisE7 remains in its closed conformation over 99% of the simulated time in atomistic molecular dynamic (MD) simulations. 9 This does not prevent O 2 from escaping to the solvent directly from the distal heme site with the putative E7 gate closed. 9 Also, 47−79% of O 2 escapes occur from the R and T quaternary states of HbA via its interior tunnels with HisE7 closed.…”

“…9 This does not prevent O 2 from escaping to the solvent directly from the distal heme site with the putative E7 gate closed. 9 Also, 47−79% of O 2 escapes occur from the R and T quaternary states of HbA via its interior tunnels with HisE7 closed. 9 Since our simulation results on HbA with HisE7 closed are at odds with the E7-gate hypothesis, we asked if opening of HisE7 would favor direct escape to the solvent from the distal heme pockets.…”

“…9 Also, 47−79% of O 2 escapes occur from the R and T quaternary states of HbA via its interior tunnels with HisE7 closed. 9 Since our simulation results on HbA with HisE7 closed are at odds with the E7-gate hypothesis, we asked if opening of HisE7 would favor direct escape to the solvent from the distal heme pockets. We found in preliminary simulations that the imidazolium ring of the protonated HisE7 spontaneously rotates out toward the solvent (Figure 1e,h) and exploit this observation here to simulate O 2 diffusion from the distal heme pockets with HisE7 in its protonated open conformation in the high-4 affinity R and low-affinity T quaternary states of HbA.…”