JBIC Journal of Biological Inorganic Chemistry

, Volume 17, Issue 8, pp 1187–1195

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

Original Paper

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.

Keywords

Multicopper oxidase Ceruloplasmin Fet3p Iron binding Iron oxidation 

Abbreviations

BHK

Baby hamster kidney

DEAE

Diethylaminoethyl

DMEM-F12

Dulbecco’s modified Eagle’s medium–Ham F12 nutrient mixture

IEC

Ion exchange chromatography

pPD

p-Phenylenediamine

rhHp

Recombinant human hephaestin

Tris

Tris(hydroxymethyl)aminomethane

Supplementary material

775_2012_932_MOESM1_ESM.pdf (37 kb)
Supplementary material 1 (PDF 36 kb)

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Copyright information

© SBIC 2012

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular Biology, Centre for Blood ResearchUniversity of British ColumbiaVancouverCanada

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