Molecular Aspects of a Robust Assay for Ferroxidase Function of Ceruloplasmin

Ceruloplasmin (Cp) is one of the most complex multicopper oxidase enzymes and plays an essential role in the metabolism of iron in mammals. Ferrous ion supplied by the ferroportin exporter is converted by Cp to ferric ion that is accepted by plasma metallo-chaperone transferrin. Study of the enzyme at the atomic and molecular level has been hampered by the lack of a suitable ferrous substrate. We have developed the classic chromophoric complex Fe^IIH_x(Tar)₂ (H₂Tar, 4-(2-thiazolylazo)resorcinol; x = 0-2; overall charge omitted) as a robust substrate for evaluation of the ferroxidase function of Cp and related enzymes. The catalysis can be followed conveniently in real time by monitoring the solution absorbance at 720 nm, a fingerprint of Fe^IIH_x(Tar)₂. The complex is oxidized to its ferric form Fe^IIIH_x(Tar)₂ via the overall reaction sequence Fe^IIH_x(Tar)₂ → Fe^II-Cp → Fe^III-Cp → Fe^IIIH_x(Tar)₂: i.e., Fe(II) is transferred formally from Fe^IIH_x(Tar)₂ to the substrate docking/oxidation (SDO) site(s) in Cp, followed by oxidation to product Fe(III) that is trapped again by the ligand. Each Tar ligand in the above bis-complex coordinates the metal center in a meridional tridentate mode involving a pH-sensitive -OH group (pK_a > 12), and this imposes rapid Fe(II) and Fe(III) transfer kinetics to facilitate the catalytic process. The formation constants of both the ferrous and ferric complexes at pH 7.0 were determined (log β₂' = 13.6 and 21.6, respectively), as well as an average dissociation constant of the SDO site(s) in Cp (log K_D' = -7.2).