Carbonmonoxy dopamine β-hydroxylase: Structural characterization by fourier transform infrared, fluorescence, and X-ray absorption spectroscopy

The carbon monoxide complex of ascorbate-reduced dopamine β-hydroxylase has been prepared and characterized by Fourier transform infrared, fluorescence, and x-ray absorption spectroscopies. CO has previously been shown to be a competitive inhibitor with respect to O₂, and binds to only one of the two copper atoms/active site (Blackburn, N. J., Pettingill, T. M., Seagraves, K. S., and Shigeta, R. T. (1990) J. Biol. Chem. 265, 15383-15386). Thus, it acts as an excellent probe of the O₂-binding site. A single C-O infrared absorption band is observed at 2089 cm^-1, shifting by 46 cm^-1 to lower energy on substitution with either ¹³C¹⁶O or ¹²C¹⁸O. The ¹³C isotope shift is reversed to the position expected for ¹²CO upon vacuum flushing with ¹²CO gas, indicating that formation of the CO adduct is a fully reversible process. Binding of the substrate tyramine does not eliminate the infrared peak but causes a 3-cm^-1 shift to lower energy. On the other hand, binding of a bifunctional inhibitor which cross-links the substrate and O₂-binding site does eliminate the CO peak. These data, in conjunction with the competitive nature of CO binding with respect to O₂, identify the CO-binding site as the O₂-binding site, and place it in close proximity to the substrate-binding site. CO-dopamine β-hydroxylase exhibits no luminescence in the visible region, suggesting a structure different from carbonmonoxy hemocyanin, and in all probability mononuclear. Analysis of extended x-ray absorption spectroscopy data is most consistent with an average coordination per Cu of 2-3 histidines, 0.5 CO, and 0.5 S atoms as ligands, and absorption edge comparisons indicates pseudo-4 coordination as the most likely geometry at each Cu(I) center. The results can be interpreted by a model involving inequivalent 4-coordination at each Cu(I) center in the CO adduct with Cu_AHis₃S ... Cu_BHis₂CO-X as the coordination most consistent with all of the data.