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

Abstract

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.

The two oxidized forms of the trinuclear Cu cluster in the multicopper oxidases and mechanism for the decay of the native intermediate

“…This in fact provided the first evidence for the presence of the trinuclear Cu clusters in biology, which was confirmed by the first crystal structure of an MCO, ascorbate oxidase (AO) (16). However, crystal structures of azide-bound AO (17), ceruloplasmin (Cp) (18), and CotA (19) later showed disagreement with spectroscopy (the three are also different from one another), indicating that the azide binding only occurs at the T3 site.…”

“…Recent model studies combined with calculations have further demonstrated that the three Cu(II) centers in the trinuclear site are all bridged by a 3 -oxo ligand (8,9). In the absence of reducing substrate, NI slowly decays to the resting enzyme, in which the one remaining O atom of the O 2 is terminally bound as OH Ϫ to the T2 site, as indicated by 18 O isotope ratio MS (IRMS) (10,11) and 17 O EPR (12) experiments. Thus, this process requires the 3 -oxo-bridged NI to undergo a large rearrangement.…”

Automated matching of high- and low-resolution structural models

“…The structure of apo human ceruloplasmin (hCP) (Zaitsev et al, 1999), containing 1046 residues, was taken as a template in simulation No 18. The apo-hCP contains three domains linked by¯exible loops freely moving in solution (Vachette, 1999).…”

In vitro studies indicate a quinone is involved in bacterial Mn(II) oxidation

“…SD21 was investigated (Table 3). Sodium azide and sodium cyanide are known to inhibit MCOs and quinoproteins (Solomon et al 1996;Zaitsev et al 1999), and high concentrations of these inhibitors did inhibit Mn(II) oxidation. Surprisingly, o-phenanthroline, a specific copper chelator and known inhibitor of Mn(II) oxidation in other species, only partially inhibited Mn(II) oxidation at high concentrations-much higher than was required to inhibit Mn(II) oxidation in partially purified preparations of Pseudomonas putida GB-1 (Okazaki et al 1997).…”