The structure of the copper sites in oxidized and reduced dopamine β-hydroxylase has been studied by extended x-ray absorption fine structure spectroscopy using a restrained refinement approach to data analysis. An histidine-rich active site has been found to be present with an average histidine coordination of between two and three histidine ligands per copper. In the oxidized protein, the data support four-coordination, involving two to three imidazole groups at 1.99 Å with additional ligands derived from water or exogenous O-donor groups at an average distance of 1.94 Å. Studies on the reduced enzyme have focused on resolving the controversy in the literature (Scott, R. A., Sullivan, R. J., De Wolfe, W. E., Dolle, R. E., and Kruse, L. I. (1988) Biochemistry 27, 5411-5417; Blumberg, W. E., Desai, P. R., Powers, L., Freedman, J. H., and Villafranca, J. J. (1989) J. Biol. Chem. 264, 6029-6032) as to whether a S/Cl scatterer is a ligand to Cu(I). Five independent samples of reduced enzyme prepared under conditions designed to probe the Cu(I) ligand environment have been measured and analyzed. All five samples gave identical spectra and could be simulated by two to three imidazoles (1.93 A) and 0.5 S/Cl (2.25 A) per Cu(I). The spectra were insensitive to the presence of added bromide or to exclusion of chloride during preparation. The results establish that the heavy atom scatterer is derived from a sulfur donor. Some evidence was found for an additional O/N scatterer at 2.6 Å in the reduced enzyme. A hypothesis for the structure of the copper sites has been proposed involving inequivalent CuA(His)3(H2O) ⋯ CuB-(His)2X(H2O) coordination in the oxidized enzyme, which upon reduction loses coordinated water and coordinates a sulfur probably from a methionine.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - Dec 5 1991|
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