Multi-copper oxidases and human iron metabolism

Abstract

Multi-copper oxidases (MCOs) are a small group of enzymes that oxidize their substrate with the concomitant reduction of dioxygen to two water molecules. Generally, multi-copper oxidases are promiscuous with regards to their reducing substrates and are capable of performing various functions in different species. To date, three multi-copper oxidases have been detected in humans--ceruloplasmin, hephaestin and zyklopen. Each of these enzymes has a high specificity towards iron with the resulting ferroxidase activity being associated with ferroportin, the only known iron exporter protein in humans. Ferroportin exports iron as Fe(2+), but transferrin, the major iron transporter protein of blood, can bind only Fe(3+) effectively. Iron oxidation in enterocytes is mediated mainly by hephaestin thus allowing dietary iron to enter the bloodstream. Zyklopen is involved in iron efflux from placental trophoblasts during iron transfer from mother to fetus. Release of iron from the liver relies on ferroportin and the ferroxidase activity of ceruloplasmin which is found in blood in a soluble form. Ceruloplasmin, hephaestin and zyklopen show distinctive expression patterns and have unique mechanisms for regulating their expression. These features of human multi-copper ferroxidases can serve as a basis for the precise control of iron efflux in different tissues. In this manuscript, we review the biochemical and biological properties of the three human MCOs and discuss their potential roles in human iron homeostasis.

Figure 1

Transport of iron in the enterocyte. Proteins are shown with abbreviated names. Transporters are shows in blue; ferriductases and ferroxidases are depicted in orange; iron storage proteins are shown in green. Fe²⁺ in the dashed circle represents labile cytosolic iron pool.

Figure 2

Mechanism of O₂ reduction to water by the multi-copper oxidases (MCOs). Broad arrows indicate the steps that take place in the catalytic cycle of the MCO. Thin arrows indicate steps that can be experimentally observed but are not part of the catalytic cycle. Cu1 and Cu2 represent type 1 and type 2 copper, respectively, Cu3a and Cu3b are type 3 copper atoms. See [97] for a full discussion.

Figure 3

(a) The ribbon diagram of human ceruloplasmin. Top view of the molecule along the pseudo-3-fold axis. (b) Side view of ceruloplasmin almost perpendicular to the pseudo-3-fold axis with the putative iron binding site. (c) Iron-binding site in domain 6 of ceruloplasmin. Residues E272, E935, H940 and D1025 represent iron ligands, residue H1026 is a ligand of type 1 copper in domain 6. Arrow shows putative electron transfer path from the iron atom to the adjacent type 1 copper atom. Figures were generated using Pymol software (PDB ID 1KCW, Schrödinger, Portland, OR, USA).

Figure 4

(a) Surface model of ceruloplasmin, top view of the molecule; predicted ligands of high-affinity iron-binding sites in domain 4 (E597, H602, D684, E971) and in domain 6 (E935, H940, D1025, E272) are shown as sticks. (b) Same as A; protein loops that cover iron-binding sites are shown as ribbons; groups of residues at the top and at the bottom represent iron-binding sites in domains 6 and 4 respectively. (c) Surface charge distribution on human ceruloplasmin. Top view of the molecule along the pseudo-3-fold axis. (d) Surface charge distribution on human ceruloplasmin. Bottom view of the molecule along the pseudo-3-fold axis. The negative and positive electrostatic potential regions are scaled from red for −76 to blue for +76 kT/e. The crystal structure of ceruloplasmin (PDB code 2J5W) was visualized with Pymol; the electrostatic map was obtained using APBS plug-in.