The copper centers of tyramine β-monooxygenase and its catalytic-site methionine variants

An X-ray absorption study

Corinna R. Hess, Judith P. Klinman, Ninian Blackburn

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Tyramine β-monooxygenase (TBM) is a member of a family of copper monooxygenases containing two noncoupled copper centers, and includes peptidylglycine monooxygenase and dopamine β-monooxygenase. In its Cu(II) form, TBM is coordinated by two to three His residues and one to two non-His O/N ligands consistent with a [CuM(His)2(OH2) 2-CuH(His)3(OH2)] formulation. Reduction to the Cu(I) state causes a change in the X-ray absorption spectroscopy (XAS) spectrum, consistent with a change to a [Cu M(His)2S(Met)-CuH(His)3] environment. Lowering the pH to 4.0 results in a large increase in the intensity of the Cu(I)-S extended X-ray absorption fine structure (EXAFS) component, suggesting a tighter Cu-S bond or the coordination of an additional sulfur donor. The XAS spectra of three variants, where the CuM Met471 residue had been mutated to His, Cys, and Asp, were examined. Significant differences from the wild-type enzyme are evident in the spectra of the reduced mutants. Although the side chains of His, Cys, and Asp are expected to substitute for Met at the CuM site, the data showed identical spectra for all three reduced variants, with no evidence for coordination of residue 471. Rather, the K-edge data suggested a modest decrease in coordination number, whereas the EXAFS indicated an average of two His residues at each Cu(I) center. These data highlight the unique role of the Met residue at the Cu M center, and pose interesting questions as to why replacement by the cuprophilic thiolate ligand leads to detectable activity whereas replacement by imidazole generates inactive TBM.

Original languageEnglish (US)
Pages (from-to)1195-1207
Number of pages13
JournalJournal of Biological Inorganic Chemistry
Volume15
Issue number8
DOIs
StatePublished - Nov 2010
Externally publishedYes

Fingerprint

Tyramine
X ray absorption
Mixed Function Oxygenases
X-Ray Absorption Spectroscopy
Methionine
Copper
Catalytic Domain
X ray absorption spectroscopy
X-Rays
Viperidae
Ligands
Sulfur
Dopamine
Enzymes

Keywords

  • Copper
  • Extended X-ray absorption fine structure
  • Tyramine β-monooxygenase
  • X-ray absorption

ASJC Scopus subject areas

  • Biochemistry
  • Inorganic Chemistry

Cite this

The copper centers of tyramine β-monooxygenase and its catalytic-site methionine variants : An X-ray absorption study. / Hess, Corinna R.; Klinman, Judith P.; Blackburn, Ninian.

In: Journal of Biological Inorganic Chemistry, Vol. 15, No. 8, 11.2010, p. 1195-1207.

Research output: Contribution to journalArticle

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abstract = "Tyramine β-monooxygenase (TBM) is a member of a family of copper monooxygenases containing two noncoupled copper centers, and includes peptidylglycine monooxygenase and dopamine β-monooxygenase. In its Cu(II) form, TBM is coordinated by two to three His residues and one to two non-His O/N ligands consistent with a [CuM(His)2(OH2) 2-CuH(His)3(OH2)] formulation. Reduction to the Cu(I) state causes a change in the X-ray absorption spectroscopy (XAS) spectrum, consistent with a change to a [Cu M(His)2S(Met)-CuH(His)3] environment. Lowering the pH to 4.0 results in a large increase in the intensity of the Cu(I)-S extended X-ray absorption fine structure (EXAFS) component, suggesting a tighter Cu-S bond or the coordination of an additional sulfur donor. The XAS spectra of three variants, where the CuM Met471 residue had been mutated to His, Cys, and Asp, were examined. Significant differences from the wild-type enzyme are evident in the spectra of the reduced mutants. Although the side chains of His, Cys, and Asp are expected to substitute for Met at the CuM site, the data showed identical spectra for all three reduced variants, with no evidence for coordination of residue 471. Rather, the K-edge data suggested a modest decrease in coordination number, whereas the EXAFS indicated an average of two His residues at each Cu(I) center. These data highlight the unique role of the Met residue at the Cu M center, and pose interesting questions as to why replacement by the cuprophilic thiolate ligand leads to detectable activity whereas replacement by imidazole generates inactive TBM.",
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AB - Tyramine β-monooxygenase (TBM) is a member of a family of copper monooxygenases containing two noncoupled copper centers, and includes peptidylglycine monooxygenase and dopamine β-monooxygenase. In its Cu(II) form, TBM is coordinated by two to three His residues and one to two non-His O/N ligands consistent with a [CuM(His)2(OH2) 2-CuH(His)3(OH2)] formulation. Reduction to the Cu(I) state causes a change in the X-ray absorption spectroscopy (XAS) spectrum, consistent with a change to a [Cu M(His)2S(Met)-CuH(His)3] environment. Lowering the pH to 4.0 results in a large increase in the intensity of the Cu(I)-S extended X-ray absorption fine structure (EXAFS) component, suggesting a tighter Cu-S bond or the coordination of an additional sulfur donor. The XAS spectra of three variants, where the CuM Met471 residue had been mutated to His, Cys, and Asp, were examined. Significant differences from the wild-type enzyme are evident in the spectra of the reduced mutants. Although the side chains of His, Cys, and Asp are expected to substitute for Met at the CuM site, the data showed identical spectra for all three reduced variants, with no evidence for coordination of residue 471. Rather, the K-edge data suggested a modest decrease in coordination number, whereas the EXAFS indicated an average of two His residues at each Cu(I) center. These data highlight the unique role of the Met residue at the Cu M center, and pose interesting questions as to why replacement by the cuprophilic thiolate ligand leads to detectable activity whereas replacement by imidazole generates inactive TBM.

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