Cytochrome b5, not superoxide anion radical, is a major reductant of indoleamine 2,3-dioxygenase in human cells

The heme protein indoleamine 2,3-dioxygenase (IDO) initiates oxidative metabolism of tryptophan along the kynurenine pathway, and this requires reductive activation of Fe3+-IDO. The current dogma is that superoxide anion radical (O-2(radical anion)) 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(radical anion). 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(radical anion) generated by xanthine oxidase modestly activated rhIDO, in reactions thatwerepreventedcompletelybysuperoxidedismutase(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(radical anion). Similarly, cellular IDO activity was unaffected by increasing SOD activity via co-expression of Cu, Zn-SOD or by increasing cellular O-2(radical anion) 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 Fe3+-IDO to Fe2+-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 activityin IDO-transfected cells. Together, our data show that cytochrome b(5) rather than O-2(radical anion) plays a major role in the activation of IDO in human cells.