On the lability and functional significance of the type 1 copper pool in ceruloplasmin

• Published in: Journal of biological inorganic chemistry Vol. 4; no. 4; pp. 441 - 446
• Main Authors: Musci, G, Fraterrigo, T Z, Calabrese, L, McMillin, D R
• Format: Journal Article
• Language: English
• Published: Germany 01.08.1999
• Subjects: Animals; Biochemistry - methods; Ceruloplasmin - chemistry; Ceruloplasmin - metabolism; Copper - chemistry; Copper - isolation & purification; Copper - metabolism; Electron Spin Resonance Spectroscopy; Sheep
• ISSN: 0949-8257; 1432-1327
• DOI: 10.1007/s007750050330

The possibility that ceruloplasmin (CP) functions as a copper transferase has fueled a continuing interest in studies of the copper release process. The principal goal of the current investigation has been to identify the most labile copper centers in sheep protein. In fact, subjecting the enzyme to a slow flux of cyanide at pH 5.2 under nitrogen in the presence of ascorbate and a phenanthroline ligand produces partially demetalated forms of the protein. By standard chromatographic techniques it is possible to isolate protein with a Cu/CP ratio of approximately 4 or approximately 5 as opposed to the native protein which has Cu/CP = 5.8. In contrast to other blue oxidases, analysis suggests that CP preferentially loses its type 1 coppers under these conditions. Thus, the spectroscopic signals from the type 1 centers exhibit a loss of intensity while the EPR signal of the type 2 copper becomes stronger. Furthermore, the Cu/CP approximately 4 and Cu/ CP approximately 5 components retain about 50% of the activity of the native protein, consistent with an intact type 2/type 3 cluster. All three type 1 copper sites appear to suffer copper loss. Reconstitution with a copper(I) reagent restores the spectroscopic properties of the native protein and 90% of the original activity. The results suggest a possible functional significance for the presence of three type 1 coppers in CP. By employing a pool of redox-active but relatively labile type 1 copper centers, the enzyme can serve as a copper donor, if necessary, without completely sacrificing its oxidase activity.