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Thr-E11 regulates O2 affinity in Cerebratulus lacteus mini-hemoglobin.

Alessandra Pesce1, Marco Nardini, Paolo Ascenzi

  • 1Department of Physics-INFM and Center for Excellence in Biomedical Research, University of Genova, Via Dodecaneso 33, 16146 Genova, Italy.

The Journal of Biological Chemistry
|May 27, 2004
PubMed
Summary
This summary is machine-generated.

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The Cerebratulus lacteus mini-hemoglobin (CerHb) exhibits high oxygen dissociation rates due to Thr-E11. Replacing Thr-E11 with Val dramatically increases oxygen affinity and alters the active site

Area of Science:

  • Biochemistry
  • Structural Biology
  • Protein Engineering

Background:

  • Mini-hemoglobin from Cerebratulus lacteus (CerHb) typically exhibits high oxygen dissociation rates and moderate affinity.
  • This unique characteristic is attributed to the presence of a polar Thr-E11 residue in its active site.

Purpose of the Study:

  • To investigate the functional and structural impact of replacing Thr-E11 with Val in CerHb.
  • To elucidate the role of the active site hydrogen bonding network in modulating globin function.

Main Methods:

  • Site-directed mutagenesis (Thr-E11 to Val).
  • Spectroscopic analysis (CO stretching frequencies).
  • X-ray crystallography (1.70 Å resolution).
  • Oxygen binding kinetics and equilibrium measurements.

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Main Results:

  • The Thr-E11 --> Val mutation decreased O2 dissociation rates 1000-fold and increased O2 affinity 130-fold.
  • Spectral shifts in heme-bound CO indicated significant changes in the active site's electrostatic environment.
  • Crystal structure revealed minimal conformational changes, with the functional alterations attributed to altered hydrogen bonding.

Conclusions:

  • The Thr-E11 residue plays a critical role in modulating CerHb's oxygen binding properties by influencing the active site hydrogen bond network.
  • Replacing Thr-E11 with Val enhances O2 affinity by enabling Tyr-B10 to form a strong hydrogen bond with the heme-bound ligand.