Substrate Effect on Flavin-Enzyme Interaction in p-Hydroxybenzoate Hydroxylase as Probed by Resonance Inverse Raman Spectroscopy

Rachelle J. Bienstock, Michael D. Morris, Lawrence M. Schopfer

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Binding between the FAD and apoprotein of p-hydroxybenzoate hydroxylase (E.C. 1.14.13.2) from Pseudomonas fluorescens has been examined by using resonance inverse Raman spectroscopy. The vibrations of the flavin chromophore have been studied in the presence of substrates (p-hydroxybenzoate, 2,4-dihydroxybenzoate, and p-aminobenzoate), inhibitors (chloride and azide), and an effector (6-hydroxynicotinate). Ternary systems involving enzyme, inhibitor, and substrate were also examined. The 1195- and 1418-cm-1 bands are significantly shifted in frequency upon binding either substrate, inhibitor, or effector. The 1163-, 1311-, and 1595-cm-1 bands shifted in the presence of inhibitor, effector, or in the ternary complex, but not in the presence of substrate, alone. The 1184-cm-1 band was affected by azide and 6-hydroxynicotinate. The 1241-cm-1 band was perturbed in the presence of 2,4-dihydroxybenzoate. Both the 1241- and 1258-cm-1 bands were shifted in the ternary complexes and in the presence of 6-hydroxynicotinate. The 1284-cm-1 band was shifted in azide. The intense 1355/1370-cm-1 band was unchanged. The 1563-cm-1 band in the free enzyme was unchanged by p-hydroxybenzoate, chloride, 6-hydroxynicotinate, and azide plus 2,4-dihydroxybenzoate but was shifted to higher wavenumbers in azide and did not appear in p-aminobenzoate, 2,4-dihydroxybenzoate, and azide plus p-hydroxybenzoate. These changes in flavin vibrational frequencies reflect conformational changes in the enzyme upon binding ligands. Hydrogen bonding between FAD at N(l), C(2)=O, N(3), C(4)=0, and amino acid chains 45–47 and 299–300 was strengthened by binding inhibitors, effectors, and substrates causing the shifts seen in the Raman spectra.

Original languageEnglish (US)
Pages (from-to)1833-1838
Number of pages6
JournalJournal of the American Chemical Society
Volume108
Issue number8
DOIs
StatePublished - Jan 1 1986

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4-Hydroxybenzoate-3-Monooxygenase
Azides
Raman Spectrum Analysis
Raman spectroscopy
Enzymes
Substrates
para-Aminobenzoates
Flavin-Adenine Dinucleotide
Chlorides
Vibrational spectra
Ternary systems
Pseudomonas fluorescens
Chromophores
Apoproteins
Vibrations (mechanical)
Enzyme Inhibitors
Hydrogen Bonding
Raman scattering
Amino acids
Vibration

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Substrate Effect on Flavin-Enzyme Interaction in p-Hydroxybenzoate Hydroxylase as Probed by Resonance Inverse Raman Spectroscopy. / Bienstock, Rachelle J.; Morris, Michael D.; Schopfer, Lawrence M.

In: Journal of the American Chemical Society, Vol. 108, No. 8, 01.01.1986, p. 1833-1838.

Research output: Contribution to journalArticle

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N2 - Binding between the FAD and apoprotein of p-hydroxybenzoate hydroxylase (E.C. 1.14.13.2) from Pseudomonas fluorescens has been examined by using resonance inverse Raman spectroscopy. The vibrations of the flavin chromophore have been studied in the presence of substrates (p-hydroxybenzoate, 2,4-dihydroxybenzoate, and p-aminobenzoate), inhibitors (chloride and azide), and an effector (6-hydroxynicotinate). Ternary systems involving enzyme, inhibitor, and substrate were also examined. The 1195- and 1418-cm-1 bands are significantly shifted in frequency upon binding either substrate, inhibitor, or effector. The 1163-, 1311-, and 1595-cm-1 bands shifted in the presence of inhibitor, effector, or in the ternary complex, but not in the presence of substrate, alone. The 1184-cm-1 band was affected by azide and 6-hydroxynicotinate. The 1241-cm-1 band was perturbed in the presence of 2,4-dihydroxybenzoate. Both the 1241- and 1258-cm-1 bands were shifted in the ternary complexes and in the presence of 6-hydroxynicotinate. The 1284-cm-1 band was shifted in azide. The intense 1355/1370-cm-1 band was unchanged. The 1563-cm-1 band in the free enzyme was unchanged by p-hydroxybenzoate, chloride, 6-hydroxynicotinate, and azide plus 2,4-dihydroxybenzoate but was shifted to higher wavenumbers in azide and did not appear in p-aminobenzoate, 2,4-dihydroxybenzoate, and azide plus p-hydroxybenzoate. These changes in flavin vibrational frequencies reflect conformational changes in the enzyme upon binding ligands. Hydrogen bonding between FAD at N(l), C(2)=O, N(3), C(4)=0, and amino acid chains 45–47 and 299–300 was strengthened by binding inhibitors, effectors, and substrates causing the shifts seen in the Raman spectra.

AB - Binding between the FAD and apoprotein of p-hydroxybenzoate hydroxylase (E.C. 1.14.13.2) from Pseudomonas fluorescens has been examined by using resonance inverse Raman spectroscopy. The vibrations of the flavin chromophore have been studied in the presence of substrates (p-hydroxybenzoate, 2,4-dihydroxybenzoate, and p-aminobenzoate), inhibitors (chloride and azide), and an effector (6-hydroxynicotinate). Ternary systems involving enzyme, inhibitor, and substrate were also examined. The 1195- and 1418-cm-1 bands are significantly shifted in frequency upon binding either substrate, inhibitor, or effector. The 1163-, 1311-, and 1595-cm-1 bands shifted in the presence of inhibitor, effector, or in the ternary complex, but not in the presence of substrate, alone. The 1184-cm-1 band was affected by azide and 6-hydroxynicotinate. The 1241-cm-1 band was perturbed in the presence of 2,4-dihydroxybenzoate. Both the 1241- and 1258-cm-1 bands were shifted in the ternary complexes and in the presence of 6-hydroxynicotinate. The 1284-cm-1 band was shifted in azide. The intense 1355/1370-cm-1 band was unchanged. The 1563-cm-1 band in the free enzyme was unchanged by p-hydroxybenzoate, chloride, 6-hydroxynicotinate, and azide plus 2,4-dihydroxybenzoate but was shifted to higher wavenumbers in azide and did not appear in p-aminobenzoate, 2,4-dihydroxybenzoate, and azide plus p-hydroxybenzoate. These changes in flavin vibrational frequencies reflect conformational changes in the enzyme upon binding ligands. Hydrogen bonding between FAD at N(l), C(2)=O, N(3), C(4)=0, and amino acid chains 45–47 and 299–300 was strengthened by binding inhibitors, effectors, and substrates causing the shifts seen in the Raman spectra.

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