Functional role of the putative iron ligands in the ferroxidase activity of recombinant human hephaestin

Abstract

Hephaestin is a multicopper ferroxidase expressed mainly in the mammalian small intestine. The ferroxidase activity of hephaestin is thought to play an important role during iron export from intestinal enterocytes and the subsequent iron loading of the blood protein transferrin, which delivers iron to the tissues. Structurally, the ectodomain of hephaestin is predicted to resemble ceruloplasmin, the soluble ferroxidase of blood. In this study, the human hephaestin ectodomain was expressed in baby hamster kidney cells and purified to electrophoretic homogeneity. Ion exchange chromatography of purified recombinant human hephaestin (rhHp) resulted in the isolation of hephaestin fractions with distinct catalytic and spectroscopic properties. The fraction of rhHp with the highest enzymatic activity also showed an enhanced molar absorptivity at 600 nm, characteristic of type 1 copper sites. Kinetic analysis revealed that rhHp possesses both high-affinity and low-affinity binding sites for ferrous iron. To investigate the role of particular residues in iron specificity of hephaestin, mutations of putative iron ligands were introduced into rhHp using site-directed mutagenesis. Kinetic analysis of ferroxidation rates of wild-type rhHp and mutants demonstrated the important roles of hephaestin residues E960 and H965 in the observed ferroxidase activity.

Physiology of Iron Metabolism

“…As previously mentioned, HCP1, which is also a ferroxidase, has also an important role during iron export from intestinal enterocytes and its subsequent loading to Tf. Structurally, the ectodomain of HCP1 resemble Cp [90]. …”

Multi-Copper Oxidases and Human Iron Metabolism

“…However, in contrast to ceruloplasmin, hephaestin was incapable of direct oxidation of biogenic amines, such as adrenaline and dopamine [130], implying a difference in biological substrate specificities between these two homologous oxidases. In addition, kinetic studies revealed that similar to ceruloplasmin, hephaestin has two types of iron-binding sites with different affinities towards ferrous iron [131]. Studies involving site-directed mutagenesis confirmed that residues E960 and H965 serve as iron ligands of a high-affinity binding site located in domain 6 of hephaestin [131].…”

“…In addition, kinetic studies revealed that similar to ceruloplasmin, hephaestin has two types of iron-binding sites with different affinities towards ferrous iron [131]. Studies involving site-directed mutagenesis confirmed that residues E960 and H965 serve as iron ligands of a high-affinity binding site located in domain 6 of hephaestin [131]. Based on homology with ceruloplasmin, the remaining ligands of this high-affinity iron-binding site are residues E300 and D996.…”

Large scale expression and purification of secreted mouse hephaestin

“…The Type I sites accept electrons from the oxidation of substrate (Fe 2+ to Fe 3+ ), and the electrons from four substrate equivalents are transferred to the trinuclear cluster where they are coupled to the four-electron reduction of O 2 to H 2 O [ 13 ]. Crystal soaking experiments further detailed two ferrous iron binding sites, each in the proximity of one of the Type I copper centers [ 14 ]. Comparative structural modeling has indicated an analogous architecture for HEPH [ 15 ].…”

“…Comparative structural modeling has indicated an analogous architecture for HEPH [ 15 ]. However, preparations of recombinant human HEPH expressed in Pichia pastoris or baby hamster kidney (BHK) cells have indicated between 3 and 4 copper ions per HEPH molecule [ 14 , 16 , 17 ]. This discrepancy could indicate that the recombinant production of HEPH leads to incomplete copper loading, or, that HEPH has a different architecture than ceruloplasmin.…”