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Structure-function studies of the Vitreoscilla hemoglobin D-region

Sang Yeol Lee1, Benjamin C Stark, Dale A Webster

  • 1Biology Division, Department of Biological, Chemical, and Physical Sciences, Illinois Institute of Technology, Chicago, IL 60616, USA.

Biochemical and Biophysical Research Communications
|March 27, 2004
PubMed

Insights

Investigating the D-region of Vitreoscilla hemoglobin (VHb) revealed key residues crucial for heme binding. This region also plays a role in VHb

Area of Science:

  • Biochemistry
  • Structural Biology
  • Protein Engineering

Background:

  • The D-region of Vitreoscilla hemoglobin (VHb), connecting helices C and E, is structurally disordered in crystal forms.
  • Understanding the functional significance of this disordered region is crucial for VHb's biological roles.

Purpose of the Study:

  • To investigate the functional importance of the disordered D-region in Vitreoscilla hemoglobin (VHb).
  • To elucidate the role of specific amino acid residues within the D-region in heme-globin interactions and protein binding.

Main Methods:

  • Site-directed mutagenesis was employed to create six VHb mutants in the D-region.
  • Spectroscopic analyses (UV-visible, FTIR) were performed on CO-liganded VHb mutants.
  • Heme/protein ratios were determined.
  • Bacterial two-hybrid screening was used to assess interactions between VHb and the flavin domain of 2,4-DNT dioxygenase.

Main Results:

  • Mutations at Asp44, Arg47, and Glu49 significantly impacted heme-globin interactions and ligand binding.
  • A structural model was proposed where the D-region forms a loop above the heme.
  • A correlation was observed between D-region perturbation and reduced interaction with the flavin domain of 2,4-DNT dioxygenase.

Conclusions:

  • Specific residues (Asp44, Arg47, Glu49) in the VHb D-region are critical for heme binding and protein interactions.
  • The D-region likely functions as a loop involved in VHb's binding to flavoproteins, suggesting a broader role in protein-protein interactions.

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