The copper sites of dopamine beta-hydroxylase: an X-ray absorption spectroscopic study.
X-ray absorption edge and extended X-ray absorption fine structure (EXAFS) spectra are reported for the Cu(I) and Cu(II) forms of bovine dopamine beta-hydroxylase (DBH; EC 1.14.17.1) and for the Cu(I) form of DBH bound either to tyramine substrate or to a multisubstrate inhibitor [Kruse, L. I., DeWolf, W. E., Jr., Chambers, P. A., & Goodhart, P. J. (1986) Biochemistry 25, 7271-7278]. A significant change in the structure of the copper sites occurs upon ascorbate-mediated reduction of Cu(II) DBH to the Cu(I) form. While the average Cu(II) site most likely consists of a square-planar array of four (N,O)-containing ligands at 1.98 A, the average Cu(I) site shows a reduction in (N,O) coordination number (from approximately 4 to approximately 2) and the addition of a S-containing ligand at 2.30 A. No change in the average Cu(I) ligand environment accompanies binding of tyramine substrate, whereas binding of a multisubstrate inhibitor, 1-(3,5-difluoro-4-hydroxybenzyl)-1H-imidazole-2(3H)-thione, causes an increase in the Cu-S coordination, consistent with inhibitor binding to the Cu(I) site through the S atom. Although excellent signal-to-noise ratio in the EXAFS spectra of ascorbate-reduced DBH facilitated analysis of outer-shell scattering for a Cu..Cu interaction, the presence of a binuclear site could not be proven or disproven due to interference from Cu...C scattering involving the carbons of imidazole ligands.
Scott
R A
RA
Department of Chemistry, University of Georgia, Athens 30602.
Sullivan
R J
RJ
DeWolf
W E
WE
Dolle
R E
RE
Kruse
L I
LI
D000313:Adrenal Medulla / Q000201:enzymology; D000818:Animals; D002417:Cattle; D003300:Copper; D004299:Dopamine beta-Hydroxylase / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D010084:Oxidation-Reduction; D013057:Spectrum Analysis; D014965:X-Rays
D003300:Copper; D004299:Dopamine beta-Hydroxylase