Modulating Distal Cavities in the α and β Subunits of Human HbA Reveals the Primary Ligand Migration Pathway

The existence of internal cavities in proteins was first discovered in sperm whale myoglobin. In 1965, Kendrew's group (1) and then, in 1985, Petsko and coworkers (2) reported crystal structures of Mb containing bound Xe. These Xe sites are found in the proximal part of the heme pocket just below pyrrole ring B (called Xe1), behind the heme next to the vinyl group of pyrrole B (Xe2), in the back of the distal pocket (Xe4), and between the EF corner and the H helix (Xe3). Later, it was demonstrated that these Xe cavities are involved in the kinetics of internal ligand movement after photolysis of Mb-ligand complexes (3–8). Molecular modeling and dynamics simulations went further and predicted that these apolar cavities may function as transient stations for ligands migrating from the solvent to the active site in Mb (9–14). However, most experimental data, including exhaustive mutagenesis mapping and time resolved X-ray crystallography, do not support these theoretical conclusions and suggest, instead, that ligands enter and escape Mb by the His(E7) gate pathway (3, 4, 15–21). Xe cavities are also present in the interior of HbI from the blood clam Scapharca inequivalvis (HbI), can trap photodissociated CO, and have been proposed to be part of the ligand migration pathway in this mollusc globin. However, blocking these docking sites with Xe, organic halides, and space-filling Phe and Trp mutations causes no appreciable change in the fraction of ligand escape from the protein. Instead, ligands appear to enter and escape the clam Hb through the distal histidine gate (22, 23). However, the His(E7) channel is not the universal pathway for ligand movement into all globins. In some bacterial truncated hemoglobins and the neuronal mini-globin from the Nemertean sea worm Cerebratulus lacteus, ligands enter the protein through an apolar tunnel between the C-terminal ends of the E and H helices (24). In Cerebratulus lacteus Hb (CerHb) either blocking the apolar tunnel with Trp mutations or filling the tunnel with Xe markedly decreases both the fraction of ligand escape to solvent and the bimolecular rate constant for ligand entry into the active site (25, 26). There is much less experimental information regarding the functional role of internal cavities in human HbA subunits. Adachi et al. (27) demonstrated, that after CO photodissociation, an electron density feature corresponding to the ligand was observed in the primary docking site, located 3.5 A away from the Fe atom and above the pyrrole C ring in α subunits of human HbA. In β subunits, a similar primary photodissociated site is occupied, and a second small ligand electron density peak is found in the back of the distal pocket, ~ 8.5 A from Fe, in a cavity circumscribed by the B10, E11, E12 and G8 amino acid side chains. Sottini et al. (28, 29) measured CO rebinding kinetics in R-state HbCO that was encapsulated in silicon hydrogels in the presence of 80% glycerol and observed two geminate phases with characteristic rebinding times of ~ 10−8 and 10−6 s. These phases were assigned to recombination from two different internal sites. Based on molecular modeling studies, the authors proposed that the slower geminate processes correspond to rebinding from more internal cavities as was observed in Mb (3). To locate these internal, apolar cavities, Savino et al. (30) crystallized deoxyHbA under high pressure of Xe gas. Several Xe atoms were modeled into the electron density with some positions corresponding spatially to the Xe4 and Xe2 cavities in Mb. Savino et al. (30) proposed that these Xe binding sites map multiple ligand migration pathways from the active site to the solvent in both subunits of human HbA. In our previous work, we attempted to shut the E7 gate in HbA by replacing the distal His with a larger Trp residue (31, 32). The bimolecular rates of CO and O2 binding to the blocked and closed Trp(E7) conformers in HbA decreased ≥ 10-fold in comparison to those for WT HbA, suggesting that ≥ 90% of ligands enter the active site via the His(E7) gate. However, we did not explore the role of distal pocket volume and internal cavities in regulating bimolecular ligand binding. In an attempt to reconcile the discrepancy between our mutagenesis work at the E7 position and the Xe binding and theoretical work of Sottini et al. (28, 29) and Savino et al. (30), respectively, we have systematically measured the effects of high Xe pressures and Phe and Trp substitutions at the internal B10, E11 and G8 positions on ligand binding to isolated α and β subunits and tetrameric HbA. The results indicate that in native HbA ligands are sequestered near the Fe atom after photodissociation, do not migrate into internal cavities, and escape and enter the distal pocket by the E7 gate, a pathway that appears to be universal for vertebrate myoglobins and red cell hemoglobins.