title: Active site of dopamine beta-hydroxylase. Comparison of enzyme derivatives containing four and eight copper atoms per tetramer using potentiometry and EPR spectroscopy.
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 beta-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 greater than 95% of the total copper, and the temperature dependence of P1/2 shows no evidence for any S = 1 state of the copper ions in the enzyme. These results suggest a lower limit of ca. 7 A 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.
authors.affiliation: Department of Chemistry, University of Manchester Institute of Science and Technology, United Kingdom.
authors.forename: N J
authors.initials: NJ
authors.lastname: Blackburn
authors.forename: M
authors.initials: M
authors.lastname: Concannon
authors.forename: S K
authors.initials: SK
authors.lastname: Shahiyan
authors.forename: F E
authors.initials: FE
authors.lastname: Mabbs
authors.forename: D
authors.initials: D
authors.lastname: Collison
mesh_terms: D000818:Animals; D001386:Azides; D001665:Binding Sites; D002417:Cattle; D003300:Copper; D004299:Dopamine beta-Hydroxylase / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D011199:Potentiometry; D011487:Protein Conformation
chemical_list: D001386:Azides; D003300:Copper; D004299:Dopamine beta-Hydroxylase