Aromatic substrate specificity of horseradish peroxidase C studied by a combined fluorescence line narrowing/energy minimization approach: The effect of localized side-chain reorganization

M. Laberge, Sz Osvath, J. Fidy

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8 Citations (Scopus)

Abstract

Horseradish peroxidase C binds a wide variety of small H-donor compounds such as benzohydroxamic acid (BHA) and 2-naphthohydroxamic acid (NHA). In this work, we use the Mg(II)-mesoporphyrin prosthetic group derivative as a spectroscopic probe of the active site and of the interaction with the substrates. We report on high-resolution fluorescence line-narrowed spectra which show that the effects of substrate binding on the electronic transitions are similar for both substrates and present data on the normal vibrational modes that are active in the vibronic spectra. Analysis of the vibrational frequencies shows that the Mg(II) ion is 5-coordinate in all cases, thus ruling out a solvent water as sixth ligand. The frequency shifts observed as a result of substrate binding are also indicative of a more rigid prosthetic group upon substrate binding. We present models for MgMP-HRP and its complexes with both substrates and compare the resulting structures on the basis of a modeling approach combining energy minimization to finite difference Poisson-Boltzmann calculations which partitions the various relative protein contributions to substrate binding. We show that the electrostatic potential of the prosthetic group is modified by the binding event. Analysis of the unbound and bound energy-minimized structures shows that the enzyme modulates substrate binding by subtle charge reorganization in the vicinity of the catalytic site and that this rearrangement is not attributable to significant secondary structure conformational changes but to side-chain reorganization.

Original languageEnglish
Pages (from-to)9226-9237
Number of pages12
JournalBiochemistry
Volume40
Issue number31
DOIs
Publication statusPublished - Aug 7 2001

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Horseradish Peroxidase
Substrate Specificity
Catalytic Domain
Fluorescence
Substrates
Static Electricity
Prosthetics
Ions
Ligands
Water
Enzymes
Proteins
Vibrational spectra
Electrostatics
Derivatives
magnesium mesoporphyrin
magnesium mesoporphyrin horseradish peroxidase
N,N-naphthaloylhydroxylamine
benzohydroxamic acid

ASJC Scopus subject areas

  • Biochemistry

Cite this

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abstract = "Horseradish peroxidase C binds a wide variety of small H-donor compounds such as benzohydroxamic acid (BHA) and 2-naphthohydroxamic acid (NHA). In this work, we use the Mg(II)-mesoporphyrin prosthetic group derivative as a spectroscopic probe of the active site and of the interaction with the substrates. We report on high-resolution fluorescence line-narrowed spectra which show that the effects of substrate binding on the electronic transitions are similar for both substrates and present data on the normal vibrational modes that are active in the vibronic spectra. Analysis of the vibrational frequencies shows that the Mg(II) ion is 5-coordinate in all cases, thus ruling out a solvent water as sixth ligand. The frequency shifts observed as a result of substrate binding are also indicative of a more rigid prosthetic group upon substrate binding. We present models for MgMP-HRP and its complexes with both substrates and compare the resulting structures on the basis of a modeling approach combining energy minimization to finite difference Poisson-Boltzmann calculations which partitions the various relative protein contributions to substrate binding. We show that the electrostatic potential of the prosthetic group is modified by the binding event. Analysis of the unbound and bound energy-minimized structures shows that the enzyme modulates substrate binding by subtle charge reorganization in the vicinity of the catalytic site and that this rearrangement is not attributable to significant secondary structure conformational changes but to side-chain reorganization.",
author = "M. Laberge and Sz Osvath and J. Fidy",
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T1 - Aromatic substrate specificity of horseradish peroxidase C studied by a combined fluorescence line narrowing/energy minimization approach

T2 - The effect of localized side-chain reorganization

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AU - Osvath, Sz

AU - Fidy, J.

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N2 - Horseradish peroxidase C binds a wide variety of small H-donor compounds such as benzohydroxamic acid (BHA) and 2-naphthohydroxamic acid (NHA). In this work, we use the Mg(II)-mesoporphyrin prosthetic group derivative as a spectroscopic probe of the active site and of the interaction with the substrates. We report on high-resolution fluorescence line-narrowed spectra which show that the effects of substrate binding on the electronic transitions are similar for both substrates and present data on the normal vibrational modes that are active in the vibronic spectra. Analysis of the vibrational frequencies shows that the Mg(II) ion is 5-coordinate in all cases, thus ruling out a solvent water as sixth ligand. The frequency shifts observed as a result of substrate binding are also indicative of a more rigid prosthetic group upon substrate binding. We present models for MgMP-HRP and its complexes with both substrates and compare the resulting structures on the basis of a modeling approach combining energy minimization to finite difference Poisson-Boltzmann calculations which partitions the various relative protein contributions to substrate binding. We show that the electrostatic potential of the prosthetic group is modified by the binding event. Analysis of the unbound and bound energy-minimized structures shows that the enzyme modulates substrate binding by subtle charge reorganization in the vicinity of the catalytic site and that this rearrangement is not attributable to significant secondary structure conformational changes but to side-chain reorganization.

AB - Horseradish peroxidase C binds a wide variety of small H-donor compounds such as benzohydroxamic acid (BHA) and 2-naphthohydroxamic acid (NHA). In this work, we use the Mg(II)-mesoporphyrin prosthetic group derivative as a spectroscopic probe of the active site and of the interaction with the substrates. We report on high-resolution fluorescence line-narrowed spectra which show that the effects of substrate binding on the electronic transitions are similar for both substrates and present data on the normal vibrational modes that are active in the vibronic spectra. Analysis of the vibrational frequencies shows that the Mg(II) ion is 5-coordinate in all cases, thus ruling out a solvent water as sixth ligand. The frequency shifts observed as a result of substrate binding are also indicative of a more rigid prosthetic group upon substrate binding. We present models for MgMP-HRP and its complexes with both substrates and compare the resulting structures on the basis of a modeling approach combining energy minimization to finite difference Poisson-Boltzmann calculations which partitions the various relative protein contributions to substrate binding. We show that the electrostatic potential of the prosthetic group is modified by the binding event. Analysis of the unbound and bound energy-minimized structures shows that the enzyme modulates substrate binding by subtle charge reorganization in the vicinity of the catalytic site and that this rearrangement is not attributable to significant secondary structure conformational changes but to side-chain reorganization.

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