The structure of horseradish peroxidase C characterized as a molten globule state after Ca2+ depletion

Krisztián Szigeti, L. Smeller, Szabolcs Osváth, Zs. Majer, J. Fidy

Research output: Contribution to journalArticle

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Abstract

The structure and activity of native horseradish peroxidase C (HRP) is stabilized by two bound Ca2+ ions. Earlier studies suggested a critical role of one of the bound Ca2+ ions but with conflicting conclusions concerning their respective importance. In this work we compare the native and totally Ca2+-depleted forms of the enzyme using pH-, pressure-, viscosity- and temperature-dependent UV absorption, CD, H/D exchange-FTIR spectroscopy and by binding the substrate benzohydroxamic acid (BHA). We report that Ca2+-depletion does not change the alpha helical content of the protein, but strongly modifies the tertiary structure and dynamics to yield a homogeneously loosened molten globule-like structure. We relate observed tertiary changes in the heme pocket to changes in the dipole orientation and coordination of a distal water molecule. Deprotonation of distal His42, linked to Asp43, itself coordinated to the distal Ca2+, perturbs a H-bonding network connecting this Ca2+ to the heme crevice that involves the distal water. The measured effects of Ca2+ depletion can be interpreted as supporting a structural role for the distal Ca2+ and for its enhanced significance in finetuning the protein structure to optimize enzyme activity.

Original languageEnglish
Pages (from-to)1965-1974
Number of pages10
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1784
Issue number12
DOIs
Publication statusPublished - Dec 2008

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Horseradish Peroxidase
Heme
Molten materials
Ions
Deprotonation
Water
Enzyme activity
Fourier Transform Infrared Spectroscopy
Enzymes
Viscosity
Spectrum Analysis
Proteins
Spectroscopy
Pressure
Molecules
Temperature
Substrates
benzohydroxamic acid

Keywords

  • Ca depletion
  • Circular dichroism
  • Conformational dynamics
  • FTIR
  • H/D exchange
  • High pressure
  • Horseradish peroxidase
  • pH effect
  • Secondary structure

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Analytical Chemistry
  • Molecular Biology

Cite this

The structure of horseradish peroxidase C characterized as a molten globule state after Ca2+ depletion. / Szigeti, Krisztián; Smeller, L.; Osváth, Szabolcs; Majer, Zs.; Fidy, J.

In: Biochimica et Biophysica Acta - Proteins and Proteomics, Vol. 1784, No. 12, 12.2008, p. 1965-1974.

Research output: Contribution to journalArticle

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