Distal heme pocket conformers of carbonmonoxy derivatives of Ascaris hemoglobin

Evidence of conformational trapping in porous sol-gel matrices

Tapan K. Das, Uri Samuni, Yu Lin, Daniel E. Goldberg, Denis L. Rousseau, Joel M. Friedman

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

We report the ligand dependence of the conformer distribution in the distal heme pocket of Ascaris suum hemoglobin (Hb) studied by resonance Raman spectroscopy. The heme-bound CO is used as a spectroscopic antenna to probe the original distribution of conformers in the dioxygen derivative of Ascaris Hb, by utilizing sol-gel encapsulation. The first step is to encapsulate the dioxygen derivative in the porous sol-gel and let the gel age, thus trapping the equilibrium conformational distribution of Ascaris dioxygen Hb. In the second step, the dioxygen ligand is replaced by CO. The sol-gel environment impedes any large scale movements, drastically slowing down the conformational relaxation triggered by the ligation change, essentially "locking in" the initial quaternary and even tertiary structure of the protein. Studying the Fe-CO frequencies of the latter sample allows evaluation of the distribution of the distal heme pocket conformers that was originally associated with the dioxygen derivative. Extending the study to the Ascaris mutants allows for examination of the effect of specific residues in the distal pocket on the conformational distribution. The choice of mutants was largely based on the anticipated variation in hydrogen bonding patterns. The results show that the sol-gel encapsulation can slow or prevent re-equilibration within the distal heme pocket of Ascaris Hb and that the distribution of distal heme pocket conformers for the CO derivative of Ascaris Hb in the sol-gel is highly dependent on the history of the sample. Additionally, we report a detailed study of the CO complex of the mutants in solution for assignment of the various heme pocket conformers, and we present a comparison of the sol-gel data with solution data. The results support a picture in which the dioxygen derivative biases the population strongly toward a tightly packed configuration that favors the network of strong hydrogen bonding interactions, and suggest that Ascaris Hb is uniquely designed for dioxygen capture.

Original languageEnglish (US)
Pages (from-to)10433-10441
Number of pages9
JournalJournal of Biological Chemistry
Volume279
Issue number11
DOIs
StatePublished - Mar 12 2004

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Polymethyl Methacrylate
Heme
Sol-gels
Gels
Carbon Monoxide
Oxygen
Derivatives
Hydrogen Bonding
Encapsulation
Hydrogen bonds
Hemoglobins
Ascaris suum
Ligands
Ascaris
Raman Spectrum Analysis
Ascaris haemoglobin
Tertiary Protein Structure
Ligation
Raman spectroscopy
History

ASJC Scopus subject areas

  • Biochemistry

Cite this

Distal heme pocket conformers of carbonmonoxy derivatives of Ascaris hemoglobin : Evidence of conformational trapping in porous sol-gel matrices. / Das, Tapan K.; Samuni, Uri; Lin, Yu; Goldberg, Daniel E.; Rousseau, Denis L.; Friedman, Joel M.

In: Journal of Biological Chemistry, Vol. 279, No. 11, 12.03.2004, p. 10433-10441.

Research output: Contribution to journalArticle

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abstract = "We report the ligand dependence of the conformer distribution in the distal heme pocket of Ascaris suum hemoglobin (Hb) studied by resonance Raman spectroscopy. The heme-bound CO is used as a spectroscopic antenna to probe the original distribution of conformers in the dioxygen derivative of Ascaris Hb, by utilizing sol-gel encapsulation. The first step is to encapsulate the dioxygen derivative in the porous sol-gel and let the gel age, thus trapping the equilibrium conformational distribution of Ascaris dioxygen Hb. In the second step, the dioxygen ligand is replaced by CO. The sol-gel environment impedes any large scale movements, drastically slowing down the conformational relaxation triggered by the ligation change, essentially {"}locking in{"} the initial quaternary and even tertiary structure of the protein. Studying the Fe-CO frequencies of the latter sample allows evaluation of the distribution of the distal heme pocket conformers that was originally associated with the dioxygen derivative. Extending the study to the Ascaris mutants allows for examination of the effect of specific residues in the distal pocket on the conformational distribution. The choice of mutants was largely based on the anticipated variation in hydrogen bonding patterns. The results show that the sol-gel encapsulation can slow or prevent re-equilibration within the distal heme pocket of Ascaris Hb and that the distribution of distal heme pocket conformers for the CO derivative of Ascaris Hb in the sol-gel is highly dependent on the history of the sample. Additionally, we report a detailed study of the CO complex of the mutants in solution for assignment of the various heme pocket conformers, and we present a comparison of the sol-gel data with solution data. The results support a picture in which the dioxygen derivative biases the population strongly toward a tightly packed configuration that favors the network of strong hydrogen bonding interactions, and suggest that Ascaris Hb is uniquely designed for dioxygen capture.",
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