An X-ray crystallographic study of the binding sites of the azide inhibitor and organic substrates to ceruloplasmin, a multi-copper oxidase in the plasma.

Ceruloplasmin is a multi-copper oxidase, which contains most of the copper present in the plasma. It is an acute-phase reactant that exhibits a two- to three-fold increase over the normal concentration of 300 microg/ml in adult plasma. However, the precise physiological role(s) of ceruloplasmin has been the subject of intensive debate and it is likely that the enzyme has a multi-functional role, including iron oxidase activity and the oxidation of biogenic amines. The three-dimensional X-ray structure of the human enzyme was elucidated in 1996 and showed that the molecule was composed of six cupredoxin-type domains arranged in a triangular array. There are six integral copper atoms per molecule (mononuclear sites in domains 2, 4 and 6 and a trinuclear site between domains 1 and 6) and two labile sites with roughly 50% occupancy. Further structural studies on the binding of metal cations by the enzyme indicated a putative mechanism for ferroxidase activity. In this paper we report medium-resolution X-ray studies (3.0-3.5 A) which locate the binding sites for an inhibitor (azide) and various substrates [aromatic diamines, biogenic amines and (+)-lysergic acid diethylamide, LSD]. The binding site of the azide moiety is topologically equivalent to one of the sites reported for ascorbate oxidase. However, there are two distinct binding sites for amine substrates: aromatic diamines bind on the bottom of domain 4 remote from the mononuclear copper site, whereas the biogenic amine series typified by serotonin, epinephrine and dopa bind in close vicinity to that utilised by cations in domain 6 and close to the mononuclear copper. These binding sites are discussed in terms of possible oxidative mechanisms. The binding site for LSD is also reported.