Altered Ligand Rebinding Kinetics Due to Distal-side Effects in Hemoglobin Chico (Lysβ66(E10) → Thr)

Celia Bonaventura,Joseph Bonaventura,Daniel Tzu-Bi Shih,E Timothy Iben,Joel Friedman

doi:10.1074/jbc.274.13.8686

Celia Bonaventura, Joseph Bonaventura + Show 3 more

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https://doi.org/10.1074/jbc.274.13.8686

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Abstract

Hb Chico is an unusual human hemoglobin variant that has lowered oxygen affinity, but unaltered cooperativity and anion sensitivity. Previous studies showed these features to be associated with distal-side heme pocket alterations that confer increased structural rigidity on the molecule and that increase water content in the beta-chain heme pocket. We report here that the extent of nanosecond geminate rebinding of oxygen to the variant and its isolated beta-chains is appreciably decreased. Structural alterations in this variant decrease its oxygen recombination rates without significantly altering rates of migration out of the heme pocket. Data analysis indicates that one or more barriers that impede rebinding of oxygen from docking sites in the heme pocket are increased, with less consequence for CO rebinding. Resonance Raman spectra show no significant alterations in spectral regions sensitive to interactions between the heme iron and the proximal histidine residue, confirming that the functional differences in the variant are due to distal-side heme pocket alterations. These effects are discussed in the context of a schematic representation of heme pocket wells and barriers that could aid the design of novel hemoglobins with altered ligand affinity without loss of the normal allosteric responses that facilitate unloading of oxygen to respiring tissues.

Highlights

Hb Chico is an unusual human hemoglobin variant that has lowered oxygen affinity, but unaltered cooperativity and anion sensitivity
These effects are discussed in the context of a schematic representation of heme pocket wells and barriers that could aid the design of novel hemoglobins with altered ligand affinity without loss of the normal allosteric responses that facilitate unloading of oxygen to respiring tissues
Increased structural rigidity of residues in the heme pocket was invoked as part of the explanation for the fact that the entire oxygen-binding curve of Hb Chico is shifted toward lower affinity

Summary

Introduction

Hb Chico is an unusual human hemoglobin variant that has lowered oxygen affinity, but unaltered cooperativity and anion sensitivity Previous studies showed these features to be associated with distal-side heme pocket alterations that confer increased structural rigidity on the molecule and that increase water content in the ␤-chain heme pocket. The substitution of Lys with Thr at position ␤66 was inferred to result in increased structural rigidity on the distal side of the heme pocket as a result of hydrogen bonding between the distal histidine of the ␤-chains and Thr␤66(E10) through a bridging water molecule This interaction would be expected to reduce any ligand-stabilizing effects associated with hydrogen bonding between the distal histidine and bound oxygen, the extent to which hydrogen bonding stabilizes bound ligands in the ␤-chain of Hb A0 is still debated. Lowered oxygen affinity is observed for both isolated ␤-chains of Hb Chico and Hb Chico tetramers, indicating that this functional change has a tertiary basis apart from any alteration of the normal quaternary equilibrium between the R- and T-states [9]

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