The state of the copper sites in human ceruloplasmin.
The state of the various, spectroscopically distinguishable copper sites (type 1, type 2, and type 3 copper) of human ceruloplasmin was investigated by electron spin resonance (ESR) spectroscopy. The ESR measurements were performed at 100 K and at X-band during the reaction of the protein with either ascorbate or with ferricyanide. A method was developed to directly measure the contribution of type 1 and type 2 copper signals to the ESR spectrum of the native protein. A signal arising from an unperturbed type 2 copper site, obtained by aerobically treating the protein with ascorbate, allowed the estimation that the number of type 2 copper centers detectable by ESR was substantially lower than unity. A fraction of type 1 copper sites was found to be in the reduced state and could be reoxidized by treatment with ferricyanide. The data obtained were consistent with the presence of three type 1 copper sites per protein molecule. Based on the experimentally determined stoichiometries, computer simulations of the ESR lineshape were carried out which confirmed the presence of three nonequivalent type 1 copper sites and of a noninteger amount of ESR-detectable type 2 copper in human ceruloplasmin.
Musci
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Center of Molecular Biology of Consiglio Nazionale delle Ricerche, University of Rome La Sapienza, Italy.
Bonaccorsi di Patti
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MC
Calabrese
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D000693:Anaerobiosis; D001205:Ascorbic Acid / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D002570:Ceruloplasmin / Q000737:chemistry* / Q000378:metabolism; D003300:Copper / Q000032:analysis* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D006801:Humans
D003300:Copper; D002570:Ceruloplasmin; D001205:Ascorbic Acid