Ceruloplasmin (Cp) is one of the Most complex multicopper oxidase enzymes, and plays an essential role in the metabolism anon in mammals. Ferrous ion, supplied by the ferroportin exporter is converted by Cp to fern( 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 subtrate. We have developed the classic chromophoric complex (FeHx)-H-II(Tar)(2).(H,Tar, 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 (FeHx)-H-II (Tar)2. The complex is oxidized to its ferric form (FeHx)-H-III(Tar)2 via, the overall reaction sequenc (FeHx)-H-II(Tar)(2), -> Fe-II Cp -> (FeHx)-H-III(Tar)(2) i.e., Fe(II) is transferred formally from (FeHx)-H-II(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 beta(2)' = 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).