Recombinant expression and initial characterization of the putative human enteric ferric reductase Dcytb.
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+, lambdamax 413.5 nm; Fe2+, lambdamax = 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 b561 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 b561 family to provide axial ligands to bound heme, indicated that these variants were able to bind just one heme group each.
Ludwiczek
Susanne
S
Department of Biochemistry and Molecular Biology and the UBC Centre for Blood Research, Life Sciences Centre, 2350 Health Sciences Mall, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Rosell
Federico I
FI
Ludwiczek
Martin L
ML
Mauk
A Grant
AG
D000818:Animals; D018938:Caco-2 Cells; D002942:Circular Dichroism; D003573:Cytochrome b Group / Q000302:isolation & purification / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004583:Electrons; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008024:Ligands; D008280:Magnetics; D051379:Mice; D050505:Mutant Proteins / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D013053:Spectrophotometry; D017186:Titrimetry
D003573:Cytochrome b Group; D008024:Ligands; D050505:Mutant Proteins; D011994:Recombinant Proteins; D010088:Oxidoreductases; C422926:CYBRD1 protein, human