Human indoleamine 2,3-dioxygenase is a catalyst of physiological heme peroxidase reactions: implications for the inhibition of dioxygenase activity by hydrogen peroxide.

The heme enzyme indoleamine 2,3-dioxygenase (IDO) is a key regulator of immune responses through catalyzing l-tryptophan (l-Trp) oxidation. Here, we show that hydrogen peroxide (H(2)O(2)) activates the peroxidase function of IDO to induce protein oxidation and inhibit dioxygenase activity. Exposure of IDO-expressing cells or recombinant human IDO (rIDO) to H(2)O(2) inhibited dioxygenase activity in a manner abrogated by l-Trp. Dioxygenase inhibition correlated with IDO-catalyzed H(2)O(2) consumption, compound I-mediated formation of protein-centered radicals, altered protein secondary structure, and opening of the distal heme pocket to promote nonproductive substrate binding; these changes were inhibited by l-Trp, the heme ligand cyanide, or free radical scavengers. Protection by l-Trp coincided with its oxidation into oxindolylalanine and kynurenine and the formation of a compound II-type ferryl-oxo heme. Physiological peroxidase substrates, ascorbate or tyrosine, enhanced rIDO-mediated H(2)O(2) consumption and attenuated H(2)O(2)-induced protein oxidation and dioxygenase inhibition. In the presence of H(2)O(2), rIDO catalytically consumed nitric oxide (NO) and utilized nitrite to promote 3-nitrotyrosine formation on IDO. The promotion of H(2)O(2) consumption by peroxidase substrates, NO consumption, and IDO nitration was inhibited by l-Trp. This study identifies IDO as a heme peroxidase that, in the absence of substrates, self-inactivates dioxygenase activity via compound I-initiated protein oxidation. l-Trp protects against dioxygenase inactivation by reacting with compound I and retarding compound II reduction to suppress peroxidase turnover. Peroxidase-mediated dioxygenase inactivation, NO consumption, or protein nitration may modulate the biological actions of IDO expressed in inflammatory tissues where the levels of H(2)O(2) and NO are elevated and l-Trp is low.

Flavivirus infection induces indoleamine 2,3-dioxygenase in human monocyte-derived macrophages via tumor necrosis factor and NF-κB.

Infection with West Nile virus (WNV) via a mosquito bite results in local viral replication in the skin, followed by viremia. Thus, tissue macrophages are ideally located to prevent the dissemination of WNV throughout the host. The current study shows that WNV infection of human monocyte-derived macrophages (MDM) results in increased WNV mRNA, protein, and infectious virions at 24 h p.i. with a decline in titer after 48 h. Concomitant with viral control was the robust induction of indoleamine 2,3-dioxygenase (IDO) and resultant metabolism of L-tryptophan (L-Trp) to kynurenine. In WNV-exposed cultures, IDO protein was induced primarily in noninfected versus viral-infected MDM. Whereas WNV infection increased the production of IFN-α, IFN-ß, and TNF, only antibody neutralization of TNF attenuated IDO expression and activity. WNV infection also activated NF-κB, and inhibition of this pathway with BMS-345541 abrogated IDO induction. Similar results were also obtained with MDM infected with the related flavivirus, Japanese encephalitis virus. Whereas IDO-mediated L-Trp metabolism can exhibit antiviral properties, inhibition of IDO activity in MDM with L-1-MT or the addition of excess L-Trp did not affect viral control. However, culturing MDM in L-Trp-deficient medium or overexpression of IDO in cells prior to infection significantly attenuated WNV replication, which was reversed by adding excess L-Trp. Together, these data support that although IDO is not required by MDM for the clearance of established viral infection, the spread of flavivirus infection is limited by IDO expressed in uninfected, neighboring cells.

The selenazal drug ebselen potently inhibits indoleamine 2,3-dioxygenase by targeting enzyme cysteine residues.

The heme enzyme indoleamine 2,3-dioxygenase (IDO) plays an important immune regulatory role by catalyzing the oxidative degradation of l-tryptophan. Here we show that the selenezal drug ebselen is a potent IDO inhibitor. Exposure of human macrophages to ebselen inhibited IDO activity in a manner independent of changes in protein expression. Ebselen inhibited the activity of recombinant human IDO (rIDO) with an apparent inhibition constant of 94 +/- 17 nM. Optical and resonance Raman spectroscopy showed that ebselen altered the active site heme of rIDO by inducing a transition of the ferric heme iron from the predominantly high- to low-spin form and by lowering the vibrational frequency of the Fe-CO stretch of the CO complex, indicating an opening of the distal heme pocket. Substrate binding studies showed that ebselen enhanced nonproductive l-tryptophan binding, while circular dichroism indicated that the drug reduced the helical content and protein stability of rIDO. Thiol labeling and mass spectrometry revealed that ebselen reacted with multiple cysteine residues of IDO. Removal of cysteine-bound ebselen with dithiothreitol reversed the effects of the drug on the heme environment and significantly restored enzyme activity. These findings indicate that ebselen inhibits IDO activity by reacting with the enzyme's cysteine residues that result in changes to protein conformation and active site heme, leading to an increase in the level of nonproductive substrate binding. This study highlights that modification of cysteine residues is a novel and effective means of inhibiting IDO activity. It also suggests that IDO is under redox control and that the enzyme represents a previously unrecognized in vivo target of ebselen.

Post-translational regulation of human indoleamine 2,3-dioxygenase activity by nitric oxide.

The heme protein indoleamine 2,3-dioxygenase (IDO) is induced by the proinflammatory cytokine interferon-gamma (IFNgamma) and plays an important role in the immune response by catalyzing the oxidative degradation of L-tryptophan (Trp) that contributes to immune suppression and tolerance. Here we examined the mechanism by which nitric oxide (NO) inhibits human IDO activity. Exposure of IFNgamma-stimulated human monocyte-derived macrophages (MDM) to NO donors had no material impact on IDO mRNA or protein expression, yet exposure of MDM or transfected COS-7 cells expressing active human IDO to NO donors resulted in reversible inhibition of IDO activity. NO also inhibited the activity of purified recombinant human IDO (rhIDO) in a reversible manner and this correlated with NO binding to the heme of rhIDO. Optical absorption and resonance Raman spectroscopy identified NO-inactivated rhIDO as a ferrous iron (Fe(II))-NO-Trp adduct. Stopped-flow kinetic studies revealed that NO reacted most rapidly with Fe(II) rhIDO in the presence of Trp. These findings demonstrate that NO inhibits rhIDO activity reversibly by binding to the active site heme to trap the enzyme as an inactive nitrosyl-Fe(II) enzyme adduct with Trp bound and O2 displaced. Reversible inhibition by NO may represent an important mechanism in controlling the immune regulatory actions of IDO.