Metal coordination influences substrate binding in horseradish peroxidase

E. Balog, R. Galántai, M. Köhler, M. Laberge, J. Fidy

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

To clarify the role of metal ion coordination in horseradish peroxidase C (HRPC), the effect of pressure and of an externally applied electric field on spectral holes was compared for both metal-free and Mg-mesoporphyrin-substituted horseradish peroxidase C (MP-HRP and MgMP-HRP), as affected by the binding of 2-naphthohydroxamic acid (NHA). The data are compared to earlier studies performed on the same derivatives. Results obtained for MP-HRP show the presence of a predominant MP tautomer, as well as that of another small population with different pocket field and isothermal compressibility (0.12 vs 0.24 GPa-1). Binding NHA induces the formation of two new almost equal populations of MP-HRP tautomer complexes and the protein compressibility in both forms is increased to 0.50 and 0.36 GPa-1. The protein structure becomes much softer than in the absence of NHA. Binding the same substrate to MgMP-HRP resulted in MgMP adopting a single conformation with no compressibility changes, while without NHA, two forms were possible. Stark effect results show charge rearrangement upon substrate binding in both cases. We propose that it is the presence of the metal that stabilizes the structure during the reorganization of the protein matrix induced by the substrate binding event. With the metal, only one conformation is adopted, without significant structural rearrangement but with charge redistribution. The dissociation constants determined for NHA binding to both derivatives and to native HRPC show that studies using mesoporphyrin and Mg-mesoporphyrin derivatives are relevant to investigating the specificity of the substrate-binding pocket in this enzyme.

Original languageEnglish
Pages (from-to)429-438
Number of pages10
JournalEuropean Biophysics Journal
Volume29
Issue number6
DOIs
Publication statusPublished - Jan 1 2000

Keywords

  • Horseradish peroxidase
  • Porphyrin Q-band splitting
  • Protein isothermal compressibility
  • Spectral hole burning
  • Stark effect

ASJC Scopus subject areas

  • Biophysics

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