The multifunctional oxidase activity of ceruloplasmin as revealed by anionbinding studies

The effect of multiple binding of azide, N-3(-), on the structural and func tional 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 oxid ase activity toward Fe(II) was essentially unaffected, while turnover param eters versus nonferrous substrates dramatically changed, with an approximat e to 100-fold enhancement of the k(cat)/K-m parameter. Chloride at physiolo gical concentration proved to behave very similarly to N-3(-) bound with hi gh affinity, in that it not only induced the spectroscopic changes previous ly interpreted in terms of an intramolecular electron transfer from reduced type 1 to type 3 copper ions [Musci, G., Bonaccorsi di Patti, M.C. & Calab rese, 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 affini ty N-3(-) or Cl-. At higher N-3(-) concentrations, optical changes of both human and chicken CP were mainly focussed on the appearance of ligand-to-me tal charge transfer bands below 500 nm, and the anion behaved as an inhibit or 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(-)/CP ratios. The data presented in this paper a re consistent with a mechanism of structural and functional modulation of C P 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 mul tifunctional oxidase.