PREPARATION AND CHARACTERIZATION OF HALF-APO DOPAMINE-BETA-HYDROXYLASE BY SELECTIVE REMOVAL OF CU-A - IDENTIFICATION OF A SULFUR LIGAND AT THE DIOXYGEN BINDING-SITE BY EXAFS AND FTIR SPECTROSCOPY

Progress has been made in determining the individual coordination of each of the copper sites (CUA and CUB) which comprise the active center in dopamine-beta-hydroxylase. Previous studies (Blackburn et al. J. Biol. Chem. 1991, 266, 23 120-27) have determined the average ligand environment per copper in the fully metalated enzyme as two to three histidines and one to two O/N donors in the Cu(II) form changing to 2-3 histidines and 0.5 sulfur donors upon reduction to the Cu(I) form. Derivatives of the Cu(I) form of DBH have been made in which CUA has been selectively removed, allowing CUB, the O-2-binding center to be studied by EXAFS and FTIR. CUB has been found to be coordinated to two histidines (Cu-N = 1.99 +/- 0.03 Angstrom), a S-donor ligand (Cu-S = 2.25 +/- 0.02 A), and a fourth, as yet Unidentified ligand X (Cu-X = 2.53 +/- 0.03 Angstrom). The FTIR spectrum of the carbonyl derivative of CUB indicates that nu(CO) (2089 cm(-1)) is identical to that found for the fully metalated enzyme, providing strong evidence that the CUB Site is not perturbed by CUA removal. EXAFS results on Cu-B-CO indicate that CO binding does not displace the S ligand but appears to displace the weakly bound ligand X. These results provide the first evidence for the involvement of sulfur ligation at the dioxygen binding site of a copper monooxygenase. Amino acid residues which could act as potential S donors are discussed, and it is suggested that a methionine is the most likely candidate. The implications of sulfur ligation on the hydroxylation mechanism are discussed.