Differences in the binding of aromatic substrates to horseradish peroxidase revealed by fluorescence line narrowing

J. Fidy, K. G. Paul, Jane M. Vanderkooi

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Abstract

The heme in horseradish peroxidase (HRP) isoenzyme C was replaced by mesoporphyrin (MP), and the binding effect of the aromatic substrates benzo- and naphthohydroxamic acid (BHA, NHA), resorcinol (RE), isomeric resorcylic acids (α-, β-, γ-RE), and hydroquinone (HQ) was studied at pH 5 by conventional and laser-excited fluorescence spectroscopy on the basis of the signal of the porphyrin. Under laser excitation at cryogenic temperatures site selection was demonstrated, and the fluorescence line narrowing data were used to characterize the HRP/substrate complexes by the inhomogeneous distribution function for the S0 ← S1 (0 ← 0) transition energy and the vibrational energies in the S1 electronic state. A comparison with ground-state vibrational energies for MP in chloroform/ether showed a downward shift in vibrational energies for S1 by ≈20 cm-1. The association characteristics of the substrates were in accordance with previous literature data indicating NHA to be of the strongest binding affinity. For BHA, spectral evidence was obtained for a second type of binding site where hydrophobic interactions with the porphyrin ring may be possible. The effect of the RE's was similar to each other, but only β-RE showed saturation. Complexation in every case caused the strong reduction of the splitting in the 0 ← 0 transition energy for the tautomeric forms of MP and an increase in the 0 ← 0 energy by 100-200 cm-1 depending on the substrate. The substrate binding also affected the phonon coupling of vibronic transitions exciting into the Δν = 927- and 976-cm-1 modes; in the latter case, the vibrational energy was also increased to 983 cm-1 for β-RE. In the same energy range, however, the transition into the Δν = 958-960-cm-1 mode was not affected by binding. Both the magnitude of the energy shifting and the change in the strength of phonon coupling gave the same relation, BHA <NHA <HQ <RE's, indicating a common conformational origin. A reduction of the fluctuational freedom of the protein chain at room temperature within the heme pocket was suggested on the basis of the reduction of the width of the inhomogeneous distribution of 0 ← 0 energies (from 60-70 to ≈30 cm-1 in case of HRP/HQ) upon substrate binding. Ways to relate the transition energy splitting and shifting effects to conformational changes are discussed by invoking the Jahn-Teller effect.

Original languageEnglish
Pages (from-to)7531-7541
Number of pages11
JournalBiochemistry
Volume28
Issue number19
Publication statusPublished - 1989

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Horseradish Peroxidase
Butylated Hydroxyanisole
Fluorescence
Phonons
Porphyrins
Substrates
Heme
Lasers
Temperature
Acids
Fluorescence Spectrometry
Chloroform
Hydrophobic and Hydrophilic Interactions
Ether
Isoenzymes
Jahn-Teller effect
Site selection
Laser excitation
Binding Sites
Fluorescence spectroscopy

ASJC Scopus subject areas

  • Biochemistry

Cite this

Differences in the binding of aromatic substrates to horseradish peroxidase revealed by fluorescence line narrowing. / Fidy, J.; Paul, K. G.; Vanderkooi, Jane M.

In: Biochemistry, Vol. 28, No. 19, 1989, p. 7531-7541.

Research output: Contribution to journalArticle

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abstract = "The heme in horseradish peroxidase (HRP) isoenzyme C was replaced by mesoporphyrin (MP), and the binding effect of the aromatic substrates benzo- and naphthohydroxamic acid (BHA, NHA), resorcinol (RE), isomeric resorcylic acids (α-, β-, γ-RE), and hydroquinone (HQ) was studied at pH 5 by conventional and laser-excited fluorescence spectroscopy on the basis of the signal of the porphyrin. Under laser excitation at cryogenic temperatures site selection was demonstrated, and the fluorescence line narrowing data were used to characterize the HRP/substrate complexes by the inhomogeneous distribution function for the S0 ← S1 (0 ← 0) transition energy and the vibrational energies in the S1 electronic state. A comparison with ground-state vibrational energies for MP in chloroform/ether showed a downward shift in vibrational energies for S1 by ≈20 cm-1. The association characteristics of the substrates were in accordance with previous literature data indicating NHA to be of the strongest binding affinity. For BHA, spectral evidence was obtained for a second type of binding site where hydrophobic interactions with the porphyrin ring may be possible. The effect of the RE's was similar to each other, but only β-RE showed saturation. Complexation in every case caused the strong reduction of the splitting in the 0 ← 0 transition energy for the tautomeric forms of MP and an increase in the 0 ← 0 energy by 100-200 cm-1 depending on the substrate. The substrate binding also affected the phonon coupling of vibronic transitions exciting into the Δν = 927- and 976-cm-1 modes; in the latter case, the vibrational energy was also increased to 983 cm-1 for β-RE. In the same energy range, however, the transition into the Δν = 958-960-cm-1 mode was not affected by binding. Both the magnitude of the energy shifting and the change in the strength of phonon coupling gave the same relation, BHA <NHA <HQ <RE's, indicating a common conformational origin. A reduction of the fluctuational freedom of the protein chain at room temperature within the heme pocket was suggested on the basis of the reduction of the width of the inhomogeneous distribution of 0 ← 0 energies (from 60-70 to ≈30 cm-1 in case of HRP/HQ) upon substrate binding. Ways to relate the transition energy splitting and shifting effects to conformational changes are discussed by invoking the Jahn-Teller effect.",
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AU - Paul, K. G.

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N2 - The heme in horseradish peroxidase (HRP) isoenzyme C was replaced by mesoporphyrin (MP), and the binding effect of the aromatic substrates benzo- and naphthohydroxamic acid (BHA, NHA), resorcinol (RE), isomeric resorcylic acids (α-, β-, γ-RE), and hydroquinone (HQ) was studied at pH 5 by conventional and laser-excited fluorescence spectroscopy on the basis of the signal of the porphyrin. Under laser excitation at cryogenic temperatures site selection was demonstrated, and the fluorescence line narrowing data were used to characterize the HRP/substrate complexes by the inhomogeneous distribution function for the S0 ← S1 (0 ← 0) transition energy and the vibrational energies in the S1 electronic state. A comparison with ground-state vibrational energies for MP in chloroform/ether showed a downward shift in vibrational energies for S1 by ≈20 cm-1. The association characteristics of the substrates were in accordance with previous literature data indicating NHA to be of the strongest binding affinity. For BHA, spectral evidence was obtained for a second type of binding site where hydrophobic interactions with the porphyrin ring may be possible. The effect of the RE's was similar to each other, but only β-RE showed saturation. Complexation in every case caused the strong reduction of the splitting in the 0 ← 0 transition energy for the tautomeric forms of MP and an increase in the 0 ← 0 energy by 100-200 cm-1 depending on the substrate. The substrate binding also affected the phonon coupling of vibronic transitions exciting into the Δν = 927- and 976-cm-1 modes; in the latter case, the vibrational energy was also increased to 983 cm-1 for β-RE. In the same energy range, however, the transition into the Δν = 958-960-cm-1 mode was not affected by binding. Both the magnitude of the energy shifting and the change in the strength of phonon coupling gave the same relation, BHA <NHA <HQ <RE's, indicating a common conformational origin. A reduction of the fluctuational freedom of the protein chain at room temperature within the heme pocket was suggested on the basis of the reduction of the width of the inhomogeneous distribution of 0 ← 0 energies (from 60-70 to ≈30 cm-1 in case of HRP/HQ) upon substrate binding. Ways to relate the transition energy splitting and shifting effects to conformational changes are discussed by invoking the Jahn-Teller effect.

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