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 journalArticlepeer-review

11 Scopus citations


Binding between the FAD and apoprotein of p-hydroxybenzoate hydroxylase (E.C. 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
Issue number8
StatePublished - 1986
Externally publishedYes

ASJC Scopus subject areas

  • Catalysis
  • General Chemistry
  • Biochemistry
  • Colloid and Surface Chemistry


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