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Involvement of the distal histidine in the low affinity exhibited by Hb Chico (Lys beta 66----Thr) and its isolated

C Bonaventura1, R Cashon, J Bonaventura

  • 1Marine Biomedical Center, Duke University Marine Laboratory, Beaufort, North Carolina 28516.

The Journal of Biological Chemistry
|December 5, 1991
PubMed

Insights

Hemoglobin Chico exhibits reduced oxygen affinity due to steric hindrance at Thr beta 66, impacting both T and R states. This variant shows altered Bohr effect and slower ligand binding kinetics.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Hematology

Background:

  • Hemoglobin (Hb) Chico, a variant with a Lys beta 66 to Thr substitution (E10), is known to have diminished oxygen affinity.
  • Previous studies indicated altered oxygen binding properties, but the precise molecular mechanisms were not fully elucidated.

Purpose of the Study:

  • To investigate the molecular basis of the reduced oxygen affinity in Hemoglobin Chico.
  • To characterize the functional consequences of the Thr beta 66 substitution on oxygen and carbon monoxide binding.
  • To explore the role of Lys beta 66 in the alkaline Bohr effect.

Main Methods:

  • P50 measurements to assess oxygen affinity.
  • Analysis of cooperativity, anion binding, and Bohr effects.
  • X-ray crystallography of the deoxy structure.
  • Kinetic studies of oxygen and carbon monoxide binding.
  • Measurement of O2/CO partition coefficient.

Main Results:

  • Hb Chico has approximately twice the P50 of Hb A, indicating significantly lower oxygen affinity.
  • Cooperativity, anion, and Bohr effects between pH 7-8 are normal, but the Bohr effect above pH 8 is reduced.
  • X-ray analysis revealed steric hindrance at Thr beta 66 in the T-state, slowing ligand binding.
  • Both T-state and R-state tetramers, as well as isolated beta chains, show lowered O2 and CO affinity.

Conclusions:

  • The Thr beta 66 substitution in Hb Chico introduces steric hindrance, reducing oxygen affinity by affecting ligand binding rates.
  • A minor role for the ionic bond of Lys beta 66 in the alkaline Bohr effect is identified.
  • The reduced oxygen affinity is a property of both T and R states and isolated beta chains.

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