The multifunctional oxidase activity of ceruloplasmin as revealed by anion binding studies

Abstract

The effect of multiple binding of azide, N 3 2 , on the structural and functional properties of ceruloplasmin (CP) has been reinvestigated by means of both spectroscopic and enzymatic techniques. High affinity binding of the anion to human CP resulted in a dramatic increase of the absorbance at 610 nm and in a concomitant decrease of the optical density at 330 nm. The oxidase activity toward Fe(II) was essentially unaffected, while turnover parameters versus nonferrous substrates dramatically changed, with an < 100-fold enhancement of the k cat /K m parameter. Chloride at physiological concentration proved to behave very similarly to N 3 2 bound with high affinity, in that it not only induced the spectroscopic changes previously interpreted in terms of an intramolecular electron transfer from reduced type 1 to type 3 copper ions [Musci, G., Bonaccorsi di Patti, M.C. & Calabrese, L. (1995) J. Protein Chem. 14, 611±617], but it also enhanced some 60-fold the k cat /K m value. A different behavior was observed with chicken CP, where a decrease at 330 nm occurred without a concomitant modification at 603 nm. The chicken enzyme was less sensitive also in terms of enzymatic activity, which was nearly unchanged in the presence of either high affinity N 3 2 or Cl 2 . At higher N 3 2 concentrations, optical changes of both human and chicken CP were mainly focussed on the appearance of ligand-to-metal charge transfer bands below 500 nm, and the anion behaved as an inhibitor of the oxidase activity versus Fe(II) as well as noniron substrates. The well known bleaching of the blue chromophore could be observed, at neutral pH, only at very high N 3 2 /CP ratios. The data presented in this paper are consistent with a mechanism of structural and functional modulation of CP by anions, that would be able to dictate the substrate specificity of the cuproprotein, and suggest the possibility that CP may act in vivo as a multifunctional oxidase.Keywords: ceruloplasmin; azide; chloride; anions; iron.Ceruloplasmin (CP), the blue oxidase of vertebrate plasma, is a complex, multidomain protein which regulates iron and copper metabolism. Blue oxidases couple the oxidation of a reducing substrate to the four-electron reduction of dioxygen to water. In the case of CP, a number of substrates, including phenols and aromatic amines can be oxidized by the protein [1], and the oxidation of p-phenylenediamine (pPD) constitutes in fact a classic assay for the enzymatic activity of CP. However, Fe(II) is by far the best substrate in vitro, in terms of both K m and V max [1] and the ferroxidase activity of CP is now considered one of the main functions of the protein in vivo [2].The crystallography of human CP has confirmed the presence of six copper ions [3]. Three mononuclear sites contain type 1`blue' ions that are characterized, in their oxidized state, by a strong absorption band at around 600 nm (1 <5000 m 21´c m 21 ) and by unusual magnetic parameters in the EPR spectrum (A k # 95 Â 10 24 cm 21 ) [4]. Type 1 sites act as the route of entry fo...

Ceruloplasmin revisited: structural and functional roles of various metal cation-binding sites

“…The multifunctional nature of oxidase activity of ceruloplasmin has also been revealed in anion-binding studies (Musci et al, 1999). In contrast to cations, the anions (azide and Cl À ) appear to act either as inhibitors or activators, thus modulating the oxidase activity of the enzyme, the effect of the anions being concentration-dependent and pH-dependent.…”

“…The presence of these centres, together with previous studies on the binding of various transition metals, biogenic amines and aromatic diamines, further emphasizes the prob-ability that ceruloplasmin is multi-functional. The multifunctional nature of oxidase activity of ceruloplasmin has also been revealed in anion-binding studies (Musci et al, 1999). In contrast to cations, the anions (azide and Cl À ) appear to act either as inhibitors or activators, thus modulating the oxidase activity of the enzyme, the effect of the anions being concentration-dependent and pH-dependent.…”

Structure to function relationships in ceruloplasmin: a 'moonlighting' protein

“…Another peculiarity of human ceruloplasmin is that the redox state of its copper sites is strongly affected by chloride [27], the most abundant anion in plasma. This anion can bind to the trinuclear cluster, probably to the T2 copper, and raise its redox potential [29] enhancing both the spectroscopic and, more importantly, the catalytic properties of the T1 sites(s) [30]. Given a measured K d of ~4 mM for the purified protein with respect to a chloride concentration in plasma of over 0.1 M, one can easily infer that circulating ceruloplasmin is under the control of this anion.…”

“…The possibility that ceruloplasmin is a regulator of biogenic amine levels in the plasma and, eventually, in the brain as outlined by these early studies, and the mode of action of ceruloplasmin in these processes, is currently controversial [97,98], due to many factors. The main problem is related to the in vitro conditions of the assays that cannot mimic in vivo conditions for the action of ceruloplasmin; poor kinetic parameters are another reason, although chloride has been shown to selectively enhance the oxidase activity of ceruloplasmin towards amine substrates [30]. The picture is complicated by the extreme reactivity of the intermediates and by the involvement of oxygen radicals in these reactions.…”

Interactions of caeruloplasmin with other proteins participating in inflammation

“…That was the first indication of CP's involvement in neurodegenerative processes. Oxidation of brain catecholamines by CP was revealed in early works [24][25][26][27], but only recently was it demonstrated that, under physiological conditions, some anions increase the activity of CP with non-iron substrates by two orders, whereas Fe 2+ oxidation remains unaffected [28]. Oxidation of brain catecholamines by CP was revealed in early works [24][25][26][27], but only recently was it demonstrated that, under physiological conditions, some anions increase the activity of CP with non-iron substrates by two orders, whereas Fe 2+ oxidation remains unaffected [28].…”