Determination of Pathways for Oxygen Binding to Human Hemoglobin A

The role of His(E7) in ligand binding to HbA was re-examined in view of discrepancies between early kinetic studies and recent structure determinations. Replacing His(E7) with Gly, Leu, Phe, and Trp causes 20- to 500-fold increases in the rates of O2 dissociation from both subunits, and FfIR spectra reveal a shift in the C-O stretching frequency peak from 1950 cm-1 to -1970 cm-1 for apolar mutations, indicating loss of a positive electrostatic field next to bound ligands. Thus, the native His(E7) side chain forms a strong hydrogen bond (- -8 kJ/mol) with bound 02 in both HbA subunits. Increasing the size of the E7 residue from Gly to Trp monotonically decreases the rate constants for CO, 02 and NO association to HbA. Substituting His(E7) with Trp also slows down opening and closing of the E7 gate. Ligand binding to the Trp(E7) subunits is markedly biphasic due to a competition between very fast recombination to an open conformer and relaxation of the indole side chain to closed or blocked forms. Crystal structures of Hb and Mb Trp(E7) mutants provide structural models for these closed and blocked conformers. In the closed state, the indole side chain fills both the E7 channel and the distal pocket, inhibiting binding to iron from any direction. In the blocked state, Trp(E7) is located in the solvent interface but still blocks entry into the E7 channel. The bimolecular rate constants for CO binding to the closed and blocked states are 0.08 IlMIS- l and 0.7IlM-1S-1, respectively, which are -100 and -lO-fold slower than average wildtype parameter (-7 IlM-1s-1). Filling potential alternative ligand pathways with xenon does not affect the rate or fraction of ligand escape from either HbA subunit. In contrast, reducing the volume of the distal pocket by space-filling mutations at the BlO, Ell and GS positions dramatically affects both geminate recombination and bimolecular ligand binding. Taken together, these results demonstrate that the E7 channel is the major pathway for ligand entry and escape in HbA and that previously proposed ligand migration routes involving Xe cavities are not functionally significant

Role of Heme Pocket Water in Allosteric Regulation of Ligand Reactivity in Human Hemoglobin

Water molecules can enter the heme pocket after the escape of ligand from myoglobins and hemoglobins, hydrogen bond with the distal histidine, and introduce steric barriers to ligand rebinding. The photodissociated CO complexes of human hemoglobin and its isolated α and β chains were subjected to spectrokinetic analysis of the effect of heme hydration on ligand rebinding. A strong coupling was observed between heme hydration and quaternary state. This coupling may contribute significantly to the 20–60-fold difference between the R- and T-state bimolecular CO binding rate constants and thus to the modulation of ligand reactivity that is the hallmark of hemoglobin allostery. Heme hydration proceeded over the course of several kinetic phases in the tetramer, including the R to T quaternary transition. An initial 150 ns hydration phase increased the R-state distal pocket water occupancy, n(w)(R), to a level similar to that of the isolated α (~60%) and β (~10%) chains, resulting in a modest barrier to ligand binding. A subsequent phase, concurrent with the first step of the R → T transition, further increased the level of heme hydration, increasing the barrier. The final phase, concurrent with the final step of the allosteric transition, brought the water occupancy of the T-state tetramer, n(w)(T), even higher and close to full occupancy in both the α and β subunits (~90%). This hydration level could present an even larger barrier to ligand binding and contribute significantly to the lower iron reactivity of the T state toward CO

Role of Heme Pocket Water in Allosteric Regulation of Ligand Reactivity in Human Hemoglobin.

Water molecules can enter the heme pockets of unliganded myoglobins and hemoglobins, hydrogen bond with the distal histidine, and introduce steric barriers to ligand binding. The spectrokinetics of photodissociated CO complexes of human hemoglobin and its isolated α and β chains were analyzed for the effect of heme hydration on ligand rebinding. A strong coupling was observed between heme hydration and quaternary state. This coupling may contribute significantly to the 20-60-fold difference between the R- and T-state bimolecular CO binding rate constants and thus to the modulation of ligand reactivity that is the hallmark of hemoglobin allostery. Heme hydration proceeded over the course of several kinetic phases in the tetramer, including the R to T quaternary transition. An initial 150 ns hydration phase increased the R-state distal pocket water occupancy, nw(R), to a level similar to that of the isolated α (∼60%) and β (∼10%) chains, resulting in a modest barrier to ligand binding. A subsequent phase, concurrent with the first step of the R → T transition, further increased the level of heme hydration, increasing the barrier. The final phase, concurrent with the final step of the allosteric transition, brought the water occupancy of the T-state tetramer, nw(T), even higher and close to full occupancy in both the α and β subunits (∼90%). This hydration level could present an even larger barrier to ligand binding and contribute significantly to the lower iron reactivity of the T state toward CO

Hemoglobin Kirklareli (α H58L), a New Variant Associated with Iron Deficiency and Increased CO Binding

International audienceMutations in hemoglobin can cause a wide range of pheno-typic outcomes, including anemia due to protein instability and red cell lysis. Uncovering the biochemical basis for these phenotypes can provide new insights into hemoglobin structure and function as well as identify new therapeutic opportunities. We report here a new hemoglobin ␣ chain variant in a female patient with mild anemia, whose father also carries the trait and is from the Turkish city of Kirklareli. Both the patient and her father had a His-58(E7) 3 Leu mutation in ␣1. Surprisingly, the patient's father is not anemic, but he is a smoker with high levels of HbCO (ϳ16%). To understand these phenotypes, we examined recombinant human Hb (rHb) Kirklareli containing the ␣ H58L replacement. Mutant ␣ subunits containing Leu-58(E7) autoxidize ϳ8 times and lose hemin ϳ200 times more rapidly than native ␣ subunits, causing the oxygenated form of rHb Kirklareli to denature very rapidly under physiological conditions. The crystal structure of rHb Kirklareli shows that the ␣ H58L replacement creates a completely apolar active site, which prevents electrostatic stabilization of bound O 2 , promotes autoxidation, and enhances hemin dissociation by inhibiting water coordination to the Fe(III) atom. At the same time, the mutant ␣ subunit has an ϳ80,000-fold higher affinity for CO than O 2 , causing it to rapidly take up and retain carbon monoxide , which prevents denaturation both in vitro and in vivo and explains the phenotypic differences between the father, who is a smoker, and his daughter

Blocking the Gate to Ligand Entry in Human Hemoglobin*

His(E7) to Trp replacements in HbA lead to markedly biphasic bimolecular CO rebinding after laser photolysis. For isolated mutant subunits, the fraction of fast phase increases with increasing [CO], suggesting a competition between binding to an open conformation with an empty E7 channel and relaxation to blocked or closed, slowly reacting states. The rate of conformational relaxation of the open state is ∼18,000 s−1 in α subunits and ∼10-fold faster in β subunits, ∼175,000 s−1. Crystal structures were determined for tetrameric α(WT)β(Trp-63) HbCO, α(Trp-58)β(WT) deoxyHb, and Trp-64 deoxy- and CO-Mb as controls. In Trp-63(E7) βCO, the indole side chain is located in the solvent interface, blocking entry into the E7 channel. Similar blocked Trp-64(E7) conformations are observed in the mutant Mb crystal structures. In Trp-58(E7) deoxy-α subunits, the indole side chain fills both the channel and the distal pocket, forming a completely closed state. The bimolecular rate constant for CO binding, k′CO, to the open conformations of both mutant Hb subunits is ∼80–90 μm−1 s−1, whereas k′CO for the completely closed states is 1000-fold slower, ∼0.08 μm−1 s−1. A transient intermediate with k′CO ≈ 0.7 μm−1 s−1 is observed after photolysis of Trp-63(E7) βCO subunits and indicates that the indole ring blocks the entrance to the E7 channel, as observed in the crystal structures of Trp(E7) deoxyMb and βCO subunits. Thus, either blocking or completely filling the E7 channel dramatically slows bimolecular binding, providing strong evidence that the E7 channel is the major pathway (≥90%) for ligand entry in human hemoglobin