A Unique Ceruloplasmin Shows Distinct Redox Potentials for the Catalytic and Electron‐Transfer Metal Sites

ABSTRACT Ceruloplasmin, a multicopper oxidase essential for copper metabolism and oxidative defense, from blood serum of domestic goats ( Capra aegagrus hircus ) has been purified to homogeneity by using three steps of column chromatography. The purified goat ceruloplasmin (gCP) protein has an estimated molecular weight of approximately 141 kDa as determined by SDS‐PAGE. ICP‐AES analysis indicates that each molecule of the protein contains 5.6 ± 0.2 atoms of copper. The optimum pH of catalysis for gCP was found to be pH 5.5. Using the Hill equation, the K m and V max values for gCP are estimated to be 0.41 ± 0.01 mM and 0.35 ± 0.01 μmol/min/mg, respectively. The enzyme has an average of 3.3 ± 0.33 substrate binding sites per molecule. Both, circular dichroism (CD) spectroscopy studies and computational analysis using PDBsum reveals that gCP is primarily composed of β‐strands distinguishing it from the α‐helix‐rich human ceruloplasmin. Also, computational analysis of gCP reveals six copper‐binding sites, composed of histidine (his), methionine (Met), and cysteine (cys). Cyclic voltammetry studies of gCP immobilized on a glassy carbon electrode indicate the presence of Type 1 and Type 2/3 copper centers within the protein identified by two unique redox potential values of +294 mV and −250 mV versus the normal hydrogen electrode (NHE), respectively. The positive redox potential site that probably acts as the electron‐transfer center had a slightly lower redox potential compared to that reported in other ceruloplasmin. The negative redox potential site of the protein possibly corresponds to the catalytic site containing trinuclear copper center proposed earlier. The redox potential of the Type 1 center aligns closely with azurin, suggesting evolutionary homology and suitability for direct electron transfer (DET). The distinct electrochemical properties of gCP may enhance applications in biofuel cells, biosensors, and other bioelectronic devices due to its efficient electron‐transfer capabilities. This study provides valuable insights into the functional versatility of ceruloplasmin across species and highlights the biotechnological potential of this unique variant.