The Thermodynamics, Kinetics, and Molecular Mechanism of Intramolecular Electron Transfer in Human Ceruloplasmin

Abstract

Ceruloplasmin is a multicopper oxidase that contains three type 1 Cu sites and a type 2/type 3 trinuclear Cu cluster. All other multicopper oxidases contain only one type 1 Cu and a trinuclear cluster. In oxidized human ceruloplasmin, one type 1 Cu site is reduced and cannot be oxidized, at least in part due at to a high reduction potential, which is not catalytically relevant. Here we have examined the thermodynamics and kinetics of electron transfer among the five redox-active Cu sites to obtain insight into the molecular mechanism of intramolecular electron transfer and the function of the additional, redox-active T1 Cu site. The redox potentials of the Cu sites of human ceruloplasmin were determined by reductive and poised potential titrations. In pH 7.0 phosphate buffer, the potentials of the type 1, type 2, and type 3 Cu sites are 448, 491, and 415 mV, respectively. Cl- binds to the trinuclear cluster and significantly increases the potentials of the type 2 and type 3 Cu, indicating that Cl- is a ph...