Preparation and Characterization of Half-Apo Dopamine-β-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 (Cu_Aand CU_B) which comprise the active center in dopamine-β-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 CU_Ahas been selectively removed, allowing Cu_B, the O₂-binding center to be studied by EXAFS and FTIR. CU_Bhas been found to be coordinated to two histidines (Cu-N = 1.99 ± 0.03 Å), a S-donor ligand (Cu-S = 2.25 ± 0.02 Å), and a fourth, as yet unidentified ligand X (Cu-X = 2.53 ± 0.03 Å). The FTIR spectrum of the carbonyl derivative of Cu_Bindicates that ν_co(2089 cm^-1) is identical to that found for the fully metalated enzyme, providing strong evidence that the Cu_Bsite is not perturbed by Cu_Aremoval. 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.