Recombinant Expression and Initial Characterization of the Putative Human Enteric Ferric Reductase Dcytb

• Published in: Biochemistry (Easton) 2008-01, Vol.47 (2), p.753-761
• Main Authors: Ludwiczek, Susanne, Rosell, Federico I, Ludwiczek, Martin L, Mauk, A. Grant
• Format: Article
• Language: English
• Subjects: Animals; Caco-2 Cells; Circular Dichroism; Cytochrome b Group - isolation & purification; Cytochrome b Group - metabolism; Electron Spin Resonance Spectroscopy; Electrons; Electrophoresis, Polyacrylamide Gel; Humans; Ligands; Magnetics; Mice; Mutant Proteins - metabolism; Oxidation-Reduction; Oxidoreductases - isolation & purification; Oxidoreductases - metabolism; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Spectrophotometry; Titrimetry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi701793a

Recent studies of mutant mice with compromised ability to absorb dietary iron have identified involvement of two integral membrane proteins in the intestinal epithelial lining in iron uptake, a divalent metal ion transporter and a ferric reductase. The current study concerns the recombinant expression, purification, and initial spectroscopic characterization of a recombinant form of the human ferric reductase that was expressed and purified as the apoprotein from Escherichia coli. Reconstitution of the recombinant protein with ferriprotoporphyrin IX produced a red product with Soret (Fe3+, λmax 413.5 nm; Fe2+, λmax = 426 nm) and visible absorption maxima indicative of bisimidazole axial coordination. This observation was confirmed by electron paramagnetic resonance and magnetic circular dichroism spectroscopy. Titration of apo-Dcytb with ferriprotoporphyrin IX was consistent with the binding of two heme groups to the protein as predicted by the phylogenetic relationship of this protein to the cytochrome b 561 family. Similar titrations and spectroscopic studies of two double variants of Dcytb, each lacking a pair of histidyl residues (H50 and H120 or H86 and H159) proposed on the basis of sequence alignment with other members of the cytochrome b 561 family to provide axial ligands to bound heme, indicated that these variants were able to bind just one heme group each.