Active site of dopamine β-hydroxylase. Comparison of enzyme derivatives containing four and eight copper atoms per tetramer using potentiometry and EPR spectroscopy

Ninian Blackburn, M. Concannon, S. K. Shahiyan, F. E. Mabbs, D. Collison

Research output: Contribution to journalArticle

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Abstract

Potentiometric titrations, continuous wave EPR, and microwave power saturation measurements have been used to examine 8-Cu and 4-Cu forms of native dopamine β-hydroxylase and its azide derivative. The formation curve for the binding of Cu2+ to the apoenzyme is best fit by assuming two independent binding sites per subunit, with pK' values of 8.90 and 7.35 at pH 5.0. On the other hand, only minor differences are observed in either continuous wave EPR spectra or power saturation behavior of the 8- and 4-Cu forms of the native enzyme or of its azide derivative. The intensity of the EPR spectra of all derivatives integrates to > 95% of the total copper, and the temperature dependence of P( 1/2 ) shows no evidence for any S = 1 state of the copper ions in the enzyme. These results suggest a lower limit of ca. 7 Å for the separation between the two copper ions per subunit and thus rule out a type 3 site in the oxidized enzyme. The data are most consistent with Cu(II) sites consisting of two or three N (imidazole) and one or two O donor ligands in the coordination sphere. The similarity in EPR spectra and power saturation of 8- and 4-Cu derivatives suggests that the difference in Cu-binding constants may be due either to differences in the identity of an axial ligand or to solvation effects in the active site.

Original languageEnglish (US)
Pages (from-to)6001-6008
Number of pages8
JournalBiochemistry
Volume27
Issue number16
StatePublished - 1988
Externally publishedYes

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Potentiometry
Mixed Function Oxygenases
Paramagnetic resonance
Copper
Dopamine
Catalytic Domain
Spectrum Analysis
Azides
Spectroscopy
Derivatives
Atoms
Enzymes
Ions
Ligands
Apoenzymes
Microwaves
Solvation
Titration
Binding Sites
Temperature

ASJC Scopus subject areas

  • Biochemistry

Cite this

Active site of dopamine β-hydroxylase. Comparison of enzyme derivatives containing four and eight copper atoms per tetramer using potentiometry and EPR spectroscopy. / Blackburn, Ninian; Concannon, M.; Shahiyan, S. K.; Mabbs, F. E.; Collison, D.

In: Biochemistry, Vol. 27, No. 16, 1988, p. 6001-6008.

Research output: Contribution to journalArticle

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abstract = "Potentiometric titrations, continuous wave EPR, and microwave power saturation measurements have been used to examine 8-Cu and 4-Cu forms of native dopamine β-hydroxylase and its azide derivative. The formation curve for the binding of Cu2+ to the apoenzyme is best fit by assuming two independent binding sites per subunit, with pK' values of 8.90 and 7.35 at pH 5.0. On the other hand, only minor differences are observed in either continuous wave EPR spectra or power saturation behavior of the 8- and 4-Cu forms of the native enzyme or of its azide derivative. The intensity of the EPR spectra of all derivatives integrates to > 95{\%} of the total copper, and the temperature dependence of P( 1/2 ) shows no evidence for any S = 1 state of the copper ions in the enzyme. These results suggest a lower limit of ca. 7 {\AA} for the separation between the two copper ions per subunit and thus rule out a type 3 site in the oxidized enzyme. The data are most consistent with Cu(II) sites consisting of two or three N (imidazole) and one or two O donor ligands in the coordination sphere. The similarity in EPR spectra and power saturation of 8- and 4-Cu derivatives suggests that the difference in Cu-binding constants may be due either to differences in the identity of an axial ligand or to solvation effects in the active site.",
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T1 - Active site of dopamine β-hydroxylase. Comparison of enzyme derivatives containing four and eight copper atoms per tetramer using potentiometry and EPR spectroscopy

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AU - Concannon, M.

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AU - Mabbs, F. E.

AU - Collison, D.

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N2 - Potentiometric titrations, continuous wave EPR, and microwave power saturation measurements have been used to examine 8-Cu and 4-Cu forms of native dopamine β-hydroxylase and its azide derivative. The formation curve for the binding of Cu2+ to the apoenzyme is best fit by assuming two independent binding sites per subunit, with pK' values of 8.90 and 7.35 at pH 5.0. On the other hand, only minor differences are observed in either continuous wave EPR spectra or power saturation behavior of the 8- and 4-Cu forms of the native enzyme or of its azide derivative. The intensity of the EPR spectra of all derivatives integrates to > 95% of the total copper, and the temperature dependence of P( 1/2 ) shows no evidence for any S = 1 state of the copper ions in the enzyme. These results suggest a lower limit of ca. 7 Å for the separation between the two copper ions per subunit and thus rule out a type 3 site in the oxidized enzyme. The data are most consistent with Cu(II) sites consisting of two or three N (imidazole) and one or two O donor ligands in the coordination sphere. The similarity in EPR spectra and power saturation of 8- and 4-Cu derivatives suggests that the difference in Cu-binding constants may be due either to differences in the identity of an axial ligand or to solvation effects in the active site.

AB - Potentiometric titrations, continuous wave EPR, and microwave power saturation measurements have been used to examine 8-Cu and 4-Cu forms of native dopamine β-hydroxylase and its azide derivative. The formation curve for the binding of Cu2+ to the apoenzyme is best fit by assuming two independent binding sites per subunit, with pK' values of 8.90 and 7.35 at pH 5.0. On the other hand, only minor differences are observed in either continuous wave EPR spectra or power saturation behavior of the 8- and 4-Cu forms of the native enzyme or of its azide derivative. The intensity of the EPR spectra of all derivatives integrates to > 95% of the total copper, and the temperature dependence of P( 1/2 ) shows no evidence for any S = 1 state of the copper ions in the enzyme. These results suggest a lower limit of ca. 7 Å for the separation between the two copper ions per subunit and thus rule out a type 3 site in the oxidized enzyme. The data are most consistent with Cu(II) sites consisting of two or three N (imidazole) and one or two O donor ligands in the coordination sphere. The similarity in EPR spectra and power saturation of 8- and 4-Cu derivatives suggests that the difference in Cu-binding constants may be due either to differences in the identity of an axial ligand or to solvation effects in the active site.

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