pH dependence of the reoxidation of p-hydroxybenzoate hydroxylase · 2,4-dihydroxybenzoate complex

A. Wessiak, Lawrence M Schopfer, V. Massey

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Abstract

Oxidation of reduced p-OH-benzoate hydroxylase · 2,4-di-OH-benzoate complex by molecular oxygen occurs in four clearly defined steps. Inclusion of azide in the reaction medium slows the rate for each step. We have found that the rates for the last three steps as well as the UV/visible absorbance spectrum for the second intermediate are markedly dependent on pH. Formation of intermediate II is base-catalyzed while its decay to intermediate III is acid-catalyzed. Formation of oxydized enzyme from intermediate III is base-catalyzed. The spectrum of intermediate II is shifted to longer wavelengths on raising the pH. A pK(a) of about 7.8 can be calculated for each of these pH-dependent transitions, except for the rate of formation of oxidized enzyme. This rate appears to be directly dependent on hydroxide ion concentration. The presence of azide does not change the observed pH dependence. These results are discussed in the context of a 4a,5-ring open flavin structure of intermediate II.

Original languageEnglish (US)
Pages (from-to)12547-12556
Number of pages10
JournalJournal of Biological Chemistry
Volume259
Issue number20
StatePublished - Dec 1 1984

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4-Hydroxybenzoate-3-Monooxygenase
Azides
Benzoate 4-Monooxygenase
Molecular oxygen
Benzoates
Enzymes
Wavelength
Oxidation
Acids
Oxygen
beta-resorcylic acid
hydroxide ion

ASJC Scopus subject areas

  • Biochemistry

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pH dependence of the reoxidation of p-hydroxybenzoate hydroxylase · 2,4-dihydroxybenzoate complex. / Wessiak, A.; Schopfer, Lawrence M; Massey, V.

In: Journal of Biological Chemistry, Vol. 259, No. 20, 01.12.1984, p. 12547-12556.

Research output: Contribution to journalArticle

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N2 - Oxidation of reduced p-OH-benzoate hydroxylase · 2,4-di-OH-benzoate complex by molecular oxygen occurs in four clearly defined steps. Inclusion of azide in the reaction medium slows the rate for each step. We have found that the rates for the last three steps as well as the UV/visible absorbance spectrum for the second intermediate are markedly dependent on pH. Formation of intermediate II is base-catalyzed while its decay to intermediate III is acid-catalyzed. Formation of oxydized enzyme from intermediate III is base-catalyzed. The spectrum of intermediate II is shifted to longer wavelengths on raising the pH. A pK(a) of about 7.8 can be calculated for each of these pH-dependent transitions, except for the rate of formation of oxidized enzyme. This rate appears to be directly dependent on hydroxide ion concentration. The presence of azide does not change the observed pH dependence. These results are discussed in the context of a 4a,5-ring open flavin structure of intermediate II.

AB - Oxidation of reduced p-OH-benzoate hydroxylase · 2,4-di-OH-benzoate complex by molecular oxygen occurs in four clearly defined steps. Inclusion of azide in the reaction medium slows the rate for each step. We have found that the rates for the last three steps as well as the UV/visible absorbance spectrum for the second intermediate are markedly dependent on pH. Formation of intermediate II is base-catalyzed while its decay to intermediate III is acid-catalyzed. Formation of oxydized enzyme from intermediate III is base-catalyzed. The spectrum of intermediate II is shifted to longer wavelengths on raising the pH. A pK(a) of about 7.8 can be calculated for each of these pH-dependent transitions, except for the rate of formation of oxidized enzyme. This rate appears to be directly dependent on hydroxide ion concentration. The presence of azide does not change the observed pH dependence. These results are discussed in the context of a 4a,5-ring open flavin structure of intermediate II.

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