Interpretation of the spectra observed during oxidation of p-hydroxybenzoate hydroxylase reconstituted with modified flavins

Lawrence M Schopfer, Albert Wessiak, Vincent Massey

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

Oxidation of reduced p-hydroxybenzoate hydroxylase by oxygen in the presence of 2,4-dihydroxybenzoate and azide proceeds via three well established intermediates. Reconstitution of the apoprotein with either 8-thiophenyl-FAD, 8-fluoro-FAD, 8-chloro-FAD, or 8-sulfonyl-FAD does not alter this sequence of events. However, the peak positions of the intermediate spectra are somewhat shifted relative to those of native enzyme. Comparison of the spectra for intermediates II and III leads to the conclusion that the spectrum for intermediate II is a composite. One component is the spectrum of an intermediate III-like species, and the other appears to be related to the substrate. The substrate component is pH-dependent, having an absorbance maximum of 386 nm (extinction, ≈6,000 M-1 cm-1) at pH 6.6 which shifts to ≈430 nm (extinction, ≈11-13,000 M-1 cm-1) at pH 9.2, with a pK of 7.9. The pH dependence for the spectrum of the substrate component combined with the pH independence of the intermediate III-like spectrum satisfactorily accounts for the pH dependence observed for intermediate II, including the fact that the high pH spectrum of native intermediate II is qualitatively quite different from that of 8-sulfonyl-FAD intermediate II.

Original languageEnglish (US)
Pages (from-to)13080-13085
Number of pages6
JournalJournal of Biological Chemistry
Volume266
Issue number20
StatePublished - Dec 1 1991

Fingerprint

4-Hydroxybenzoate-3-Monooxygenase
Flavins
Flavin-Adenine Dinucleotide
Oxidation
Substrates
Apoproteins
Azides
Oxygen
Composite materials
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Interpretation of the spectra observed during oxidation of p-hydroxybenzoate hydroxylase reconstituted with modified flavins. / Schopfer, Lawrence M; Wessiak, Albert; Massey, Vincent.

In: Journal of Biological Chemistry, Vol. 266, No. 20, 01.12.1991, p. 13080-13085.

Research output: Contribution to journalArticle

@article{e3b07529a6ba4582afa6bcf5cc2af87f,
title = "Interpretation of the spectra observed during oxidation of p-hydroxybenzoate hydroxylase reconstituted with modified flavins",
abstract = "Oxidation of reduced p-hydroxybenzoate hydroxylase by oxygen in the presence of 2,4-dihydroxybenzoate and azide proceeds via three well established intermediates. Reconstitution of the apoprotein with either 8-thiophenyl-FAD, 8-fluoro-FAD, 8-chloro-FAD, or 8-sulfonyl-FAD does not alter this sequence of events. However, the peak positions of the intermediate spectra are somewhat shifted relative to those of native enzyme. Comparison of the spectra for intermediates II and III leads to the conclusion that the spectrum for intermediate II is a composite. One component is the spectrum of an intermediate III-like species, and the other appears to be related to the substrate. The substrate component is pH-dependent, having an absorbance maximum of 386 nm (extinction, ≈6,000 M-1 cm-1) at pH 6.6 which shifts to ≈430 nm (extinction, ≈11-13,000 M-1 cm-1) at pH 9.2, with a pK of 7.9. The pH dependence for the spectrum of the substrate component combined with the pH independence of the intermediate III-like spectrum satisfactorily accounts for the pH dependence observed for intermediate II, including the fact that the high pH spectrum of native intermediate II is qualitatively quite different from that of 8-sulfonyl-FAD intermediate II.",
author = "Schopfer, {Lawrence M} and Albert Wessiak and Vincent Massey",
year = "1991",
month = "12",
day = "1",
language = "English (US)",
volume = "266",
pages = "13080--13085",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "20",

}

TY - JOUR

T1 - Interpretation of the spectra observed during oxidation of p-hydroxybenzoate hydroxylase reconstituted with modified flavins

AU - Schopfer, Lawrence M

AU - Wessiak, Albert

AU - Massey, Vincent

PY - 1991/12/1

Y1 - 1991/12/1

N2 - Oxidation of reduced p-hydroxybenzoate hydroxylase by oxygen in the presence of 2,4-dihydroxybenzoate and azide proceeds via three well established intermediates. Reconstitution of the apoprotein with either 8-thiophenyl-FAD, 8-fluoro-FAD, 8-chloro-FAD, or 8-sulfonyl-FAD does not alter this sequence of events. However, the peak positions of the intermediate spectra are somewhat shifted relative to those of native enzyme. Comparison of the spectra for intermediates II and III leads to the conclusion that the spectrum for intermediate II is a composite. One component is the spectrum of an intermediate III-like species, and the other appears to be related to the substrate. The substrate component is pH-dependent, having an absorbance maximum of 386 nm (extinction, ≈6,000 M-1 cm-1) at pH 6.6 which shifts to ≈430 nm (extinction, ≈11-13,000 M-1 cm-1) at pH 9.2, with a pK of 7.9. The pH dependence for the spectrum of the substrate component combined with the pH independence of the intermediate III-like spectrum satisfactorily accounts for the pH dependence observed for intermediate II, including the fact that the high pH spectrum of native intermediate II is qualitatively quite different from that of 8-sulfonyl-FAD intermediate II.

AB - Oxidation of reduced p-hydroxybenzoate hydroxylase by oxygen in the presence of 2,4-dihydroxybenzoate and azide proceeds via three well established intermediates. Reconstitution of the apoprotein with either 8-thiophenyl-FAD, 8-fluoro-FAD, 8-chloro-FAD, or 8-sulfonyl-FAD does not alter this sequence of events. However, the peak positions of the intermediate spectra are somewhat shifted relative to those of native enzyme. Comparison of the spectra for intermediates II and III leads to the conclusion that the spectrum for intermediate II is a composite. One component is the spectrum of an intermediate III-like species, and the other appears to be related to the substrate. The substrate component is pH-dependent, having an absorbance maximum of 386 nm (extinction, ≈6,000 M-1 cm-1) at pH 6.6 which shifts to ≈430 nm (extinction, ≈11-13,000 M-1 cm-1) at pH 9.2, with a pK of 7.9. The pH dependence for the spectrum of the substrate component combined with the pH independence of the intermediate III-like spectrum satisfactorily accounts for the pH dependence observed for intermediate II, including the fact that the high pH spectrum of native intermediate II is qualitatively quite different from that of 8-sulfonyl-FAD intermediate II.

UR - http://www.scopus.com/inward/record.url?scp=0025772963&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025772963&partnerID=8YFLogxK

M3 - Article

VL - 266

SP - 13080

EP - 13085

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 20

ER -