Investigation of the anomalous spectroscopic features of the copper sites in chicken ceruloplasmin: Comparison to human ceruloplasmin

Chicken ceruloplasmin has been previously reported to display a number of k ey differences relative to human ceruloplasmin: a lower copper content and a lack of a type 2 copper signal by electron paramagnetic resonance (EPR) s pectroscopy. We have studied the copper sites of chicken ceruloplasmin in o rder to probe the origin of these differences, focusing on two forms of the enzyme: "resting" (as isolated by a fast, one-step procedure) and "peroxid e-oxidized''. From X-ray absorption, EPR, and UV/visible absorption spectro scopies, we have shown that all of the copper sites are oxidized in peroxid e-oxidized chicken ceruloplasmin and that none of the type 1 copper sites d isplay the EPR features typical for type 1 copper sites that lack an axial methionine. In the resting form, the type 2 copper center is reduced. Upon oxidation, it does not appear in the EPR spectrum at 77 K, but it can be ob served by using magnetic susceptibility, EPR at similar to 8 K, and magneti c circular dichroism spectroscopy. It displays unusually fast relaxation, i ndicative of coupling with the adjacent type 3 copper pair of the trinuclea r copper cluster. From reductive titrations, we have found that the reducti on potential of the type 2 center is higher than those of the other copper sites, thus explaining why it is reduced in the resting form. These results provide new insight into the nature of the additional type 1 copper sites and the redox distribution among copper sites in the different ceruloplasmi ns relative to other multicopper oxidases.