Effective Simulations of Gas Diffusion Through Kinetically Accessible Tunnels in Multisubunit Proteins: O₂ Pathways and Escape Routes in T-state Deoxyhemoglobin

Abstract

The diffusion of small gases to special binding sites within polypeptide matrices pivotally defines the biochemical specificity and reactivity of proteins. We investigate here explicit O(2) diffusion in adult human hemoglobin (HbA) as a case study employing the recently developed temperature-controlled locally enhanced sampling (TLES) method and vary the parameters to greatly increase the simulation efficiency. The method is carefully validated against standard molecular dynamics (MD) simulations and available experimental structural and kinetic data on ligand diffusion in T-state deoxyHbA. The methodology provides a viable alternative approach to traditional MD simulations and/or potential of mean force calculations for: (i) characterizing kinetically accessible diffusion tunnels and escape routes for light ligands in porous proteins; (ii) very large systems when realistic simulations require the inclusion of multiple subunits of a protein; and (iii) proteins that access short-lived conformations relative to the simulation time. In the case of T-state deoxyHbA, we find distinct ligand diffusion tunnels consistent with the experimentally observed disparate Xe cavities in the α- and β-subunits. We identify two distal barriers including the distal histidine (E7) that control access to the heme. The multiple escape routes uncovered by our simulations call for a review of the current popular hypothesis on ligand escape from hemoglobin. Larger deviations from the crystal structure during simulated diffusion in isolated α- and β-subunits highlight the dampening effects of subunit interactions and the importance of including all subunits of multisubunit proteins to map realistic kinetically accessible diffusion tunnels and escape routes.

Hydrophobic Effect Drives Oxygen Uptake in Myoglobin via Histidine E7

“…Bossa et al (61) and Maragliano et al (29) report the suspected opening of the His-E7 supporting the histidine gate hypothesis. On the other hand, some studies in both Hb and Mb report no opening motion of the His-E7 and raise doubt about the histidine gate hypothesis (32,62). We believe that the difference between the results that lead to opposing conclusions may depend only on the tautomeric form of His-E7 used.…”

“…A high barrier for ligand exit through the E7 channel was later reported by Maragliano et al (29), where the HID state was used, supporting our computed free energy profile. The counterintuitive result where ligand escaped through the E7 pathway when the histidine is in the closed conformation has recently been observed for Hb, although it was interpreted as evidence against the histidine gate hypothesis (32). By considering free energy profiles for all the protonation/conformational states of His-E7, we show that although ligand entry may occur in the closed His-E7 conformation, the presence of a deeper free energy well in the open conformation enhances the uptake of oxygen, lending new support for the histidine gate hypothesis.…”

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

“…Applying this approach, we mapped a network of O 2 diffusion tunnels in T leading from the distal heme sites to the solvent via multiple escape routes 12 that emanate mainly from O 2 docking sites within the tunnels. These sites in T 12 and in the isolated α-subunit 15 overlap the Xe cavities uncovered upon crystallization of HbA under Xe. 16 Similar cavities are 4 found in myoglobin (Mb), 17 a monomeric globin structurally related to HbA.…”

“…We performed standard MD simulations of the protein structures without ligands over 32 ns and PCA projections of the structures reveal that both R and R2 sample conformations close to their crystal structures whereas T gradually transitions to RR2/R (Figure 1a), consistent with its documented low conformational stability. 12,23 In contrast, during multiple 2-ns trajectories all states with TLES O 2 copies firmly represent their respective crystal subspaces ( Figure 1b). Hence, we sampled O 2 trajectories within HbA only during short 2 ns simulations.…”

“…Using a series of 2-ns TLES simulations, we found a tunnel network in T that is consistent with the results of standard MD simulations. 12,15,26 From principal component analysis (PCA) of their C α and heme atoms, de Groot and coworkers, 23 reported that the quaternary structure of T, R and R2 can be characterized by projections onto two principal components (PC1, PC2, Figure 2). The T→R transition involves linear motions mainly along PC1 (Figure 1), which correspond to rotation of the relatively rigid α 1 β 1 and α 2 β 2 heterodimers with respect to each other and results in significant changes at the heterodimer interfaces.…”