SPECTROSCOPIC AND MAGNETIC STUDIES OF HUMAN CERULOPLASMIN - IDENTIFICATION OF A REDOX-INACTIVE REDUCED TYPE-1 COPPER SITE

Ceruloplasmin is unique among the multicopper oxidases in that in addi tion to the usual copper stoichiometry of one Type 1 copper site and a Type 2/Type 3 trinuclear copper cluster, it contains two other Type 1 sites. This assignment of copper sites, based on copper quantitation, sequence alignment, and crystallography, is difficult to reconcile wi th the observed spectroscopy. Furthermore, some chemical or spectrosco pic differences in ceruloplasmin have been reported depending on the m ethod of purification. We have studied the resting (as isolated by a f ast, one-step procedure) and peroxide-oxidized forms of human cerulopl asmin. Using a combination of X-ray absorption spectroscopy, a chemica l assay, magnetic susceptibility, electron paramagnetic resonance spec troscopy, and absorption spectroscopy, we have determined that peroxid e-oxidized ceruloplasmin contains one permanently reduced Type 1 site. This site is shown to have a reduction potential of similar to 1.0 V. Thus, one of the additional Type 1 sites in ceruloplasmin cannot be c atalytically relevant in the form of the enzyme studied. Furthermore, the resting form of the enzyme contains an additional reducing equival ent, which is distributed among the remaining five copper sites as exp ected from their relative potentials. This may indicate that the resti ng form of ceruloplasmin in plasma under aerobic conditions is a four- electron oxidized form, which is consistent with its function in the f our-electron reduction of dioxygen to water.