Blocking the Gate to Ligand Entry in Human Hemoglobin

Abstract

His(E7) to Trp replacements inHemoglobins represent a very diverse protein family with members occurring in all six kingdoms of life (1). The functions of these proteins differ significantly, ranging from oxygen storage and transport, NO dioxygenation, and nitrite reduction to sensing intracellular levels of diatomic gases for transcriptional regulation and chemotaxis (2). The central chemical events for these functions are movement of ligands into the distal portion of the heme pocket, internal bond formation with the heme iron, and electrostatic stabilization or steric hindrance of the bound ligand by surrounding active site amino acids. Although the quantum mechanical details of bond formation remain to be resolved, there is general agreement on the biophysical mechanisms governing steric hindrance and hydrogen bonding between amino acid side chains and the bound ligand (3-10). In contrast, the pathways for ligands movement from solvent through the protein and into the active site are still under debate.As recently reviewed by Elber (11), molecular dynamics simulations and other computational approaches have almost uniformly suggested that there are multiple routes for ligand entry into and escape from the active sites of hemoglobins and myoglobins (12-27). Many of these pathways are coincident with apolar cavities, which have been identified as xenon docking sites (16, 17, 28 -30). However, as also pointed out by Elber (11), there is little or no direct experimental evidence in support of multiple pathways (31, 32). In the cases of mammalian myoglobins, human hemoglobin, and HbI from Scapharca inaequivalvis, almost all of the experimental evidence suggests that Ն75% of ligands enter and exit the distal pocket through a transient channel between the heme propionates, which is produced by outward rotation of the distal histidine (His(E7)). 2Although ligands do migrate into internal cavities immediately after photodissociation, they appear to return to the distal pocket and escape through the E7 gate (33-41).The structures of the active sites and the E7 channels in Mb and the subunits of human HbA are very similar, and thus analogous mechanisms for ligand binding are inferred. Until very recently, there were little experimental and computational data regarding the pathways of ligand migration into HbA. Mouawad et al. (15) observed the formation of transient cavities in the ␣ and ␤ subunits of human HbA during simulations of the T to R conformational change and postulated these cavities could allow ligands to diffuse through the globin matrix. Sottini et al. (16,17) used molecular modeling approaches to find potential xenon binding cavities in human HbA and suggested that ligands could use these apolar voids as pathways to enter or escape the active site. Savino et al. (30) reported crystal structures of Tyr(B10)/Gln(E7) deoxyHbA with xenon atoms partially occupying the sites identified in molecular modeling experiments. Thus, if only the theoretical and structural litera-* This work was supported, in whole or...

Hydrophobic Effect Drives Oxygen Uptake in Myoglobin via Histidine E7

“…Such alternative migration pathways have been observed in other globins, such as in the Mycobacterium tuberculosis truncated Hb N (58 -60) and in the Cerebratulus lacteus neuronal mini-globin (18), in which very clear apolar tunnels appear to be the main routes for ligand migration and not the E7 channel. In mammalian Mbs and Hbs, however, extensive experimental evidence has pointed to the E7 channel as the principal avenue for ligand migration (8,11,12,18). Our calculated free energy profiles for both the open and closed His-E7 conformational states also lend considerable support to the E7 channel being a principal ligand migration route in Mb.…”

“…These results also show that the Trp-E7 and Ala-E7 Mb mutants are two useful limiting cases for evaluating the E7 pathway (8,12). Association rate constants for ligand binding to Mb also suggest a pH dependence in the opening of the His-E7 gate because both kЈ O2 and kЈ CO increase 2-fold upon lowering the pH from 7.0 to 4.6 (13,14).…”

“…We should note that the new configuration adopted by Trp-E7 was previously observed in the crystal structures of the ␣ Trp-E7 mutant subunit of human recombinant deoxyHbA containing wild-type ␤ subunits (Fig. 4, right panel) (Protein Data Bank code 3NMM) (12). Both of the observed Trp-E7 conformations result in steric hindrance of the E7 pathway to the heme, as opposed to both Ala-E7 and the open His-E7 conformation (Fig.…”

Structures and Oxygen Affinities of Crystalline Human Hemoglobin C (β6 Glu→Lys) in the R and R2 Quaternary Structures

“…By contrast, so far there has been no crystallographic evidence for an opened ligand channel in human hemoglobin ␣ subunits. This is probably because the ␣ distal pocket is not so flexible as the ␤ one, as predicted by the effects of mutagenesis (28,29) and ligand size (31). Our data on R2-COHbC now suggest that the distal His(E7) in one of the ␣ subunits (␣2) is rather disordered but appears to rotate out of the distal pocket even at pH 7.6 ( Fig.…”

“…However, this disagreement cannot be considered significant because the distal His(E7) side chains are not so well ordered in the ␣2, ␤1, and ␤2 subunits in R2-COHbC, as indicated by the lack of well defined electron density map, whereas all other heme peripheral residues in R2-COHbC and those in R-COHbC (including His␣58 and His␤63) are well ordered. The observed dynamic behavior of His(E7) is not surprising because mutagenesis and kinetics studies show that the motilities of the distal His(E7) side chains are essential for opening a major channel for ligand entry in both hemoglobin subunits (28,29). Also, crystallographic evidence for the His(E7) gate has recently been obtained for a new relaxed state of hemoglobin A at pH 6.4 (referred to here as RR3; PDB code 3D17), which shows a rotation of the ␤ distal His(E7) out of the distal pocket, creating a direct channel to the bulk solvent (30).…”