Functional characterization of human duodenal cytochrome b (Cybrd1): Redox properties in relation to iron and ascorbate metabolism.
Duodenal cytochrome b (Dcytb or Cybrd1) is an iron-regulated protein, highly expressed in the duodenal brush border membrane. It has ferric reductase activity and is believed to play a physiological role in dietary iron absorption. Its sequence identifies it as a member of the cytochrome b(561) family. A His-tagged construct of human Dcytb was expressed in insect Sf9 cells and purified. Yields of protein were increased by supplementation of the cells with 5-aminolevulinic acid to stimulate heme biosynthesis. Quantitative analysis of the recombinant Dcytb indicated two heme groups per monomer. Site-directed mutagenesis of any of the four conserved histidine residues (His 50, 86, 120 and 159) to alanine resulted in much diminished levels of heme in the purified Dcytb, while mutation of the non-conserved histidine 33 had no effect on the heme content. This indicates that those conserved histidines are heme ligands, and that the protein cannot stably bind heme if any of them is absent. Recombinant Dcytb was reduced by ascorbate under anaerobic conditions, the extent of reduction being 67% of that produced by dithionite. It was readily reoxidized by ferricyanide. EPR spectroscopy showed signals from low-spin ferriheme, consistent with bis-histidine coordination. These comprised a signal at gmax=3.7 corresponding to a highly anisotropic species, and another at gmax=3.18; these species are similar to those observed in other cytochromes of the b561 family, and were reducible by ascorbate. In addition another signal was observed in some preparations at gmax=2.95, but this was unreactive with ascorbate. Redox titrations indicated an average midpoint potential for the hemes in Dcytb of +80 mV+/-30 mV; the data are consistent with either two hemes at the same potential, or differing in potential by up to 60 mV. These results indicate that Dcytb is similar to the ascorbate-reducible cytochrome b561 of the adrenal chromaffin granule, though with some differences in midpoint potentials of the hemes.
Oakhill
Jonathan S
JS
Nutritional Sciences Research Division, School of Biomedical and Health Sciences, King's College London, London, SE1 9NH, UK.
Marritt
Sophie J
SJ
Gareta
Elena Garcia
EG
Cammack
Richard
R
McKie
Andrew T
AT
D000622:Aminolevulinic Acid / Q000378:metabolism; D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular; D003573:Cytochrome b Group / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004386:Duodenum / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D005292:Ferricyanides / Q000378:metabolism; D005822:Genetic Vectors; D006418:Heme / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D006801:Humans; D007501:Iron / Q000378:metabolism*; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011199:Potentiometry; D011994:Recombinant Proteins / Q000378:metabolism; D014161:Transduction, Genetic
D003573:Cytochrome b Group; D005292:Ferricyanides; D008024:Ligands; D011994:Recombinant Proteins; C007931:hexacyanoferrate III; D006418:Heme; D006639:Histidine; D000622:Aminolevulinic Acid; D007501:Iron; D010088:Oxidoreductases; C422926:CYBRD1 protein, human; D001205:Ascorbic Acid