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Role of Copper in Thermal Stability of Human Ceruloplasmin

Erik Sedlák ^*, Gabriel oldák  and Pernilla Wittung-Stafshede ^*  

^* Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251; Laboratorium für Biochemie, Universität Bayreuth, D-95440 Bayreuth, Germany; Keck Center for Structural Computational Biology, Rice University, Houston, Texas 77251; and Department of Chemistry, Rice University, Houston, Texas 77251

Correspondence: Address reprint requests to P. Wittung-Stafshede, Tel.: 713-348-4076; Fax: 713-348-5154; E-mail: pernilla{at}rice.edu.

Human ceruloplasmin (CP) is a multicopper oxidase essential for normal iron homeostasis. The protein has six domains with one type-1 copper in each of domains 2, 4, and 6; the remaining coppers form a catalytic trinuclear cluster at the interface between domains 1 and 6. To assess the role of the coppers in CP thermal stability, we have probed the thermal unfolding process as a function of scan rate of holo- and apo-forms using several detection methods (circular dichroism, aromatic and 8-anilino-naphthalene-1-sulfonic acid fluorescence, visible absorption, activity, and differential scanning calorimetry). Both species of CP undergo irreversible thermal reactions to denatured states with significant residual structure. For identical scan rates, the thermal midpoint appears at temperatures 15–20° higher for the holo- as compared with the apo- form. The thermal data for both forms were fit by a mechanistic model involving two consecutive, irreversible steps (N I D). The holo-intermediate, I, has lost one oxidized type-1 copper and secondary structure in at least one domain; however, the trinuclear copper cluster remains intact as it is functional in oxidase activity. The activation parameters obtained from the fits to the thermal transitions were used to assess the kinetic stability of apo- and holo-CP at physiological temperatures (i.e., at 37°C). It emerges that native CP (i.e., with six coppers) is rather unstable and converts to I in <1 day at 37°C. Nonetheless, this form remains intact for more than 2 weeks and may thus be a biologically relevant state of CP in vivo. In contrast, apo-CP unfolds rapidly: the denatured state is reached in <2 days at 37°C.