Oxidation-reduction potential studies on p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens

Gary Williamson, Dale E. Edmondson, Franz Müller

Research output: Contribution to journalArticleResearchpeer-review

Abstract

The oxidation-reduction potential of p-hydroxybenzoate hydrolylase (4-hydroxybenzoate, NADPH: oxygen oxidoreductase (3-hydroxylating), EC 1.14.13.2) from Pseudomonas fluorescens has been measured in the presence and absence of p-hydroxybenzoate using spectrocoulometry. The native enzyme demonstrated a two-electron midpoint potential of -129 mV during the initial reductive titration. The midpoint potential observed during subsequent oxidative and reductive titrations was -152 mV. This marked hysteresis is proposed to arise from the oxidation and reduction of the known air-sensitive thiol group on the enzyme (Van Berkel, W.J.H. and Müller, F. (1987) Eur. J. Biochem. 167, 35-46). Redox titrations of the enzyme in the presence of substrate showed a two-electron midpoint potential of -177 mV. No spectral or electrochemical evidence for the thermodynamic stabilization of any flavin semiquinone was observed in the titrations performed. These data show that the affinity of the apoenzyme for the hydroquinone form of FAD is 150-fold greater than for the oxidized flavin and that the substrate is bound to the reduced enzyme with a 3-fold lower affinity than to the oxidized enzyme. These data are consistent with the view that the stimulatory effect of substrate binding on the rate of enzyme reduction by NADPH is due to the respective geometries of the bound FAD and NADPH rather than to a large perturbation of the oxidation-reduction potential of the bound flavin coenzyme.

Original languageEnglish
Pages (from-to)258-262
Number of pages5
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Volume953
Issue numberC
DOIs
Publication statusPublished - 1 Jan 1988
Externally publishedYes

Keywords

  • (P. fluorescens)
  • 4-Hydroxybenzoate 3-monooxygenase
  • Flavoprotein hydroxylase
  • Oxidation-reduction potential
  • p-Hydroxybenzoate hydroxylase

Cite this

@article{1cc2d79c7ada4ec3a87c4b2661d4f9b9,
title = "Oxidation-reduction potential studies on p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens",
abstract = "The oxidation-reduction potential of p-hydroxybenzoate hydrolylase (4-hydroxybenzoate, NADPH: oxygen oxidoreductase (3-hydroxylating), EC 1.14.13.2) from Pseudomonas fluorescens has been measured in the presence and absence of p-hydroxybenzoate using spectrocoulometry. The native enzyme demonstrated a two-electron midpoint potential of -129 mV during the initial reductive titration. The midpoint potential observed during subsequent oxidative and reductive titrations was -152 mV. This marked hysteresis is proposed to arise from the oxidation and reduction of the known air-sensitive thiol group on the enzyme (Van Berkel, W.J.H. and M{\"u}ller, F. (1987) Eur. J. Biochem. 167, 35-46). Redox titrations of the enzyme in the presence of substrate showed a two-electron midpoint potential of -177 mV. No spectral or electrochemical evidence for the thermodynamic stabilization of any flavin semiquinone was observed in the titrations performed. These data show that the affinity of the apoenzyme for the hydroquinone form of FAD is 150-fold greater than for the oxidized flavin and that the substrate is bound to the reduced enzyme with a 3-fold lower affinity than to the oxidized enzyme. These data are consistent with the view that the stimulatory effect of substrate binding on the rate of enzyme reduction by NADPH is due to the respective geometries of the bound FAD and NADPH rather than to a large perturbation of the oxidation-reduction potential of the bound flavin coenzyme.",
keywords = "(P. fluorescens), 4-Hydroxybenzoate 3-monooxygenase, Flavoprotein hydroxylase, Oxidation-reduction potential, p-Hydroxybenzoate hydroxylase",
author = "Gary Williamson and Edmondson, {Dale E.} and Franz M{\"u}ller",
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Oxidation-reduction potential studies on p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. / Williamson, Gary; Edmondson, Dale E.; Müller, Franz.

In: Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular, Vol. 953, No. C, 01.01.1988, p. 258-262.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Oxidation-reduction potential studies on p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens

AU - Williamson, Gary

AU - Edmondson, Dale E.

AU - Müller, Franz

PY - 1988/1/1

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N2 - The oxidation-reduction potential of p-hydroxybenzoate hydrolylase (4-hydroxybenzoate, NADPH: oxygen oxidoreductase (3-hydroxylating), EC 1.14.13.2) from Pseudomonas fluorescens has been measured in the presence and absence of p-hydroxybenzoate using spectrocoulometry. The native enzyme demonstrated a two-electron midpoint potential of -129 mV during the initial reductive titration. The midpoint potential observed during subsequent oxidative and reductive titrations was -152 mV. This marked hysteresis is proposed to arise from the oxidation and reduction of the known air-sensitive thiol group on the enzyme (Van Berkel, W.J.H. and Müller, F. (1987) Eur. J. Biochem. 167, 35-46). Redox titrations of the enzyme in the presence of substrate showed a two-electron midpoint potential of -177 mV. No spectral or electrochemical evidence for the thermodynamic stabilization of any flavin semiquinone was observed in the titrations performed. These data show that the affinity of the apoenzyme for the hydroquinone form of FAD is 150-fold greater than for the oxidized flavin and that the substrate is bound to the reduced enzyme with a 3-fold lower affinity than to the oxidized enzyme. These data are consistent with the view that the stimulatory effect of substrate binding on the rate of enzyme reduction by NADPH is due to the respective geometries of the bound FAD and NADPH rather than to a large perturbation of the oxidation-reduction potential of the bound flavin coenzyme.

AB - The oxidation-reduction potential of p-hydroxybenzoate hydrolylase (4-hydroxybenzoate, NADPH: oxygen oxidoreductase (3-hydroxylating), EC 1.14.13.2) from Pseudomonas fluorescens has been measured in the presence and absence of p-hydroxybenzoate using spectrocoulometry. The native enzyme demonstrated a two-electron midpoint potential of -129 mV during the initial reductive titration. The midpoint potential observed during subsequent oxidative and reductive titrations was -152 mV. This marked hysteresis is proposed to arise from the oxidation and reduction of the known air-sensitive thiol group on the enzyme (Van Berkel, W.J.H. and Müller, F. (1987) Eur. J. Biochem. 167, 35-46). Redox titrations of the enzyme in the presence of substrate showed a two-electron midpoint potential of -177 mV. No spectral or electrochemical evidence for the thermodynamic stabilization of any flavin semiquinone was observed in the titrations performed. These data show that the affinity of the apoenzyme for the hydroquinone form of FAD is 150-fold greater than for the oxidized flavin and that the substrate is bound to the reduced enzyme with a 3-fold lower affinity than to the oxidized enzyme. These data are consistent with the view that the stimulatory effect of substrate binding on the rate of enzyme reduction by NADPH is due to the respective geometries of the bound FAD and NADPH rather than to a large perturbation of the oxidation-reduction potential of the bound flavin coenzyme.

KW - (P. fluorescens)

KW - 4-Hydroxybenzoate 3-monooxygenase

KW - Flavoprotein hydroxylase

KW - Oxidation-reduction potential

KW - p-Hydroxybenzoate hydroxylase

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U2 - 10.1016/0167-4838(88)90033-7

DO - 10.1016/0167-4838(88)90033-7

M3 - Article

VL - 953

SP - 258

EP - 262

JO - Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology

JF - Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology

SN - 0167-4838

IS - C

ER -