Resonance Raman Studies of the Protocatechuate 3, 4-Dioxygenase from Brevibacterium Fuscum

Daniel C.T. Siu, Larry Que, Allen M. Orville, John D Lipscomb, Douglas H Ohlendorf

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27 Citations (Scopus)

Abstract

Resonance Raman studies of the protocatechuate 3, 4-dioxygenase (PCD) from Brevibacterium fuscum have been carried out to take advantage of the high iron-site homogeneity of this enzyme. Native uncomplexed PCD exhibits individual resonance-enhanced υCO and δCH vibrations for the two tyrosinates coordinated to the active site iron center, which can be assigned to a particular residue by their excitation profilés. Of the two υCO features observed at 1254 and 1266 cm-1, only the latter is upshifted (to 1272 cm-1) when H2O is replaced by D2O. Similarly the 1254-cm-1 feature is unaffected, while the 1266-cm-1 feature is Shifted to ~1290 cm-1 when inhibitors such as phenolates or terephthalate bind to the active site. These observed shifts can be rationalized by the presence of hydrogen-bonding interactions with solvent in the active site cavity, which are modulated by D2O and eliminated upon inhibitor binding. Examination of the PCD crystal structure suggests that the axial tyrosine can be hydrogen bonded in the uncomplexed enzyme to water molecules present in the substrate binding pocket. The equatorial tyrosine may also be hydrogen bonded but to solvent molecules which are trapped in a pocket inaccessible to bulk solvent. These studies allow for the first time the association of particular Raman spectroscopic features, i.e., the υCO's at 1254 and 1266 cm-1, with the equatorial and axial tyrosine residues in the PCD active site, respectively; they lay the groundwork for further Raman studies on catalytically important species to determine the roles these tyrosine residues may play in the PCD reaction cycle.

Original languageEnglish (US)
Pages (from-to)10443-10448
Number of pages6
JournalBiochemistry
Volume31
Issue number43
DOIs
StatePublished - Feb 1 1992

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Protocatechuate-3,4-Dioxygenase
Brevibacterium
Tyrosine
Catalytic Domain
Carbon Monoxide
Hydrogen
Iron
Molecules
Enzymes
Hydrogen Bonding
Vibration
Vibrations (mechanical)
Hydrogen bonds
Crystal structure
Association reactions
Water
Substrates

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Resonance Raman Studies of the Protocatechuate 3, 4-Dioxygenase from Brevibacterium Fuscum. / Siu, Daniel C.T.; Que, Larry; Orville, Allen M.; Lipscomb, John D; Ohlendorf, Douglas H.

In: Biochemistry, Vol. 31, No. 43, 01.02.1992, p. 10443-10448.

Research output: Contribution to journalArticle

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abstract = "Resonance Raman studies of the protocatechuate 3, 4-dioxygenase (PCD) from Brevibacterium fuscum have been carried out to take advantage of the high iron-site homogeneity of this enzyme. Native uncomplexed PCD exhibits individual resonance-enhanced υCO and δCH vibrations for the two tyrosinates coordinated to the active site iron center, which can be assigned to a particular residue by their excitation profil{\'e}s. Of the two υCO features observed at 1254 and 1266 cm-1, only the latter is upshifted (to 1272 cm-1) when H2O is replaced by D2O. Similarly the 1254-cm-1 feature is unaffected, while the 1266-cm-1 feature is Shifted to ~1290 cm-1 when inhibitors such as phenolates or terephthalate bind to the active site. These observed shifts can be rationalized by the presence of hydrogen-bonding interactions with solvent in the active site cavity, which are modulated by D2O and eliminated upon inhibitor binding. Examination of the PCD crystal structure suggests that the axial tyrosine can be hydrogen bonded in the uncomplexed enzyme to water molecules present in the substrate binding pocket. The equatorial tyrosine may also be hydrogen bonded but to solvent molecules which are trapped in a pocket inaccessible to bulk solvent. These studies allow for the first time the association of particular Raman spectroscopic features, i.e., the υCO's at 1254 and 1266 cm-1, with the equatorial and axial tyrosine residues in the PCD active site, respectively; they lay the groundwork for further Raman studies on catalytically important species to determine the roles these tyrosine residues may play in the PCD reaction cycle.",
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