title: Cytochrome b5, not superoxide anion radical, is a major reductant of indoleamine 2,3-dioxygenase in human cells.
abstract: The heme protein indoleamine 2,3-dioxygenase (IDO) initiates oxidative metabolism of tryptophan along the kynurenine pathway, and this requires reductive activation of Fe(3+)-IDO. The current dogma is that superoxide anion radical (O(2)(*-)) is responsible for this activation, based largely on previous work employing purified rabbit IDO and rabbit enterocytes. We have re-investigated this role of O(2)(*-) using purified recombinant human IDO (rhIDO), rabbit enterocytes that constitutively express IDO, human endothelial cells, and monocyte-derived macrophages treated with interferon-gamma to induce IDO expression, and two cell lines transfected with the human IDO gene. Both potassium superoxide and O(2)(*-) generated by xanthine oxidase modestly activated rhIDO, in reactions that were prevented completely by superoxide dismutase (SOD). In contrast, SOD mimetics had no effect on IDO activity in enterocytes and interferon-gamma-treated human cells, despite significantly decreasing cellular O(2)(*-) Similarly, cellular IDO activity was unaffected by increasing SOD activity via co-expression of Cu,Zn-SOD or by increasing cellular O(2)(*-) via treatment of cells with menadione. Other reductants, such as tetrahydrobiopterin, ascorbate, and cytochrome P450 reductase, were ineffective in activating cellular IDO. However, recombinant human cytochrome b(5) plus cytochrome P450 reductase and NADPH reduced Fe(3+)-IDO to Fe(2+)-IDO and activated rhIDO in a reconstituted system, a reaction inhibited marginally by SOD. Additionally, short interfering RNA-mediated knockdown of microsomal cytochrome b(5) significantly decreased IDO activity in IDO-transfected cells. Together, our data show that cytochrome b(5) rather than O(2)(*-) plays a major role in the activation of IDO in human cells.
authors.affiliation: Centre for Vascular Research and Molecular Immunopathology Unit, Bosch Institute and Discipline of Pathology, School of Medical Sciences, University of Sydney, Sydney, New South Wales 2006, Australia.
authors.forename: Ghassan J
authors.initials: GJ
authors.lastname: Maghzal
authors.forename: Shane R
authors.initials: SR
authors.lastname: Thomas
authors.forename: Nicholas H
authors.initials: NH
authors.lastname: Hunt
authors.forename: Roland
authors.initials: R
authors.lastname: Stocker
mesh_terms: D000818:Animals; D016466:CHO Cells; D002417:Cattle; D006224:Cricetinae; D003412:Cricetulus; D015786:Cytochromes b5 / Q000378:metabolism* / Q000494:pharmacology; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology; D020895:Enterocytes / Q000187:drug effects / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D005486:Flavin Mononucleotide / Q000494:pharmacology; D006801:Humans; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase / Q000378:metabolism*; D007371:Interferon-gamma / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects; D011817:Rabbits; D011994:Recombinant Proteins / Q000494:pharmacology; D019163:Reducing Agents / Q000378:metabolism* / Q000494:pharmacology; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism / Q000494:pharmacology; D014162:Transfection
chemical_list: D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D011994:Recombinant Proteins; D019163:Reducing Agents; D013481:Superoxides; D005486:Flavin Mononucleotide; D007371:Interferon-gamma; D015786:Cytochromes b5; D013482:Superoxide Dismutase