Human Indoleamine Research Articles

Substrate binding in human indoleamine 2,3-dioxygenase 1: A spectroscopic analysis

Human indoleamine 2,3-dioxygenase (hIDO1) is a heme enzyme that catalyzes the oxidative cleavage of the L-tryptophan indole ring. As increased levels of hIDO1 expression in tumor cells correlate with a poor prognosis for surviving several cancer types, hIDO1 has become an appealing drug target for cancer therapy. However, detailed structural knowledge of the catalytically active complex is necessary to eb able to design de novo inhibitors selective for hIDO1. Here we have applied Fourier transform infrared (FTIR) and nanosecond time-resolved optical spectroscopy to hIDO1 variants with modified heme pocket structures to identify important amino acid residues that stabilize the substrate in the active site. A cluster of small side chain residues at positions 260–265 ensures structural flexibility of the binding site. Thr379 and Arg231 are key residues acting in concert to bind the substrate. Thr379 is the final residue of a disordered loop; the neighboring Gly380, however, is still visible in the X-ray structure of the substrate-free protein, 20Å away from the heme iron. Therefore, large-scale conformational changes are necessary to bring Thr379 close to the substrate. The use of substrate analogs further reveals that an indole-like side chain with two aromatic rings and L-stereoisomery at the Cα are required for high affinity binding.

TMIC-26. EXPLORATION OF TRYPTOPHAN METABOLISM VIA THE KYNURENINE PATHWAY IN HUMAN GLIOMAS

The kynurenine pathway (KP) is responsible for a majority of the catabolism of the essential amino acid tryptophan, and recently was implicated in the pathophysiology of human gliomas. The KP generates various active metabolites, is the main source of the essential co-factor nicotinamide adenine dinucleotide (NAD+), and plays a pivotal role in promoting anti-tumor immune suppression through depleting local tryptophan and causing T-cell growth arrest. Kynurenine, a KP metabolite, was recently discovered to be an endogenous ligand of the aryl hydrocarbon receptor (AHR), a transcription factor associated with carcinogenesis. We hypothesized that higher-grade gliomas will have a more dysregulated KP, and therefore sought to examine the potential differences in KP expression in low- versus high-grade gliomas. Active tumor tissue was acquired immediately following microsurgical resection, and the tumor was dissociated into a single-cell suspension for performance of in vitro experiments. Immunohistochemical staining of Grade II, III, and IV glioma (glioblastoma: GBM) patient tumor sections with antibodies specific for: human indoleamine 2,3-dioxygenase (IDO) 1; IDO2; tryptophan 2,3-dioxgyenase (TDO2); kynureninase; kynurenine 3-monooxygenase (KMO); and AHR. Tissue sections were scored from 0-3 based on staining intensity. Patient-derived tumor cell cultures were treated with commercially available small molecule inhibitors for IDO1 (epacadostat), IDO2 (chloroquine), TDO2 (680C91), KMO (Ro61-8048), and AHR (CH-223191) for 48-hours. GBM tissues show significantly (p<0.001, ANOVA) higher levels of KP enzymes and AHR compared to Grade II and Grade III tissues. In vitro drug treatment showed markedly diminished cell proliferation via MTT assay in response to chloroquine and CH-223191. The increased staining of KP enzymes, as well as the diminished proliferation with the inhibitors, suggests that the KP plays a role in malignant gliomas, and may offer novel therapeutic targets for a patient population with highly limited treatment options.

Establishment of a human indoleamine 2, 3-dioxygenase 2 (hIDO2) bioassay system and discovery of tryptanthrin derivatives as potent hIDO2 inhibitors

As an analogue of IDO1 (indoleamine 2, 3-dioxygenase 1), the well-known new therapeutic target, IDO2 is receiving increased attention. Herein, the expression and purification of recombinant human IDO2 (hIDO2) and the establishment of a hIDO2 bioassay system on both enzymatic and cellular levels are described. Nine tryptanthrin derivatives were screened for potential hIDO2 inhibitory activities, and their Ki values, enzymatic and cellular IC50 values, as well as the types of inhibition were measured. The typtanthrin derivatives 5i, 5c and 5d (especially 5i) were found to be potent hIDO2 inhibitors with superior efficiency far better than that of the most frequently-used inhibitor L-1-MT. Two ultimate purposes of the present study have been achieved: establishing an IDO2 bioassay system and screening novel IDO2 inhibitors that can be used (directly or with some modifications) for potential therapeutic applications.

Human placental indoleamine 2,3-dioxygenase-1 is restricted to vascular endothelial and glandular epithelial expression by 5 weeks’ gestation

Human placental indoleamine 2,3-dioxygenase-1 is restricted to vascular endothelial and glandular epithelial expression by 5 weeks’ gestation

Human placental indoleamine 2,3-dioxygenase-1 is restricted to vascular endothelial and glandular epithelial expression by 5 weeks’ gestation

Human placental indoleamine 2,3-dioxygenase-1 is restricted to vascular endothelial and glandular epithelial expression by 5 weeks’ gestation

Structural Study of a Flexible Active Site Loop in Human Indoleamine 2,3-Dioxygenase and Its Functional Implications.

Human indoleamine 2,3-dioxygenase catalyzes the oxidative cleavage of tryptophan to N-formyl kynurenine, the initial and rate-limiting step in the kynurenine pathway. Additionally, this enzyme has been identified as a possible target for cancer therapy. A 20-amino acid protein segment (the JK loop), which connects the J and K helices, was not resolved in the reported hIDO crystal structure. Previous studies have shown that this loop undergoes structural rearrangement upon substrate binding. In this work, we apply a combination of replica exchange molecular dynamics simulations and site-directed mutagenesis experiments to characterize the structure and dynamics of this protein region. Our simulations show that the JK loop can be divided into two regions: the first region (JK loop(C)) displays specific and well-defined conformations and is within hydrogen bonding distance of the substrate, while the second region (JK loop(N)) is highly disordered and exposed to the solvent. The peculiar flexible nature of JK loop(N) suggests that it may function as a target for post-translational modifications and/or a mediator for protein-protein interactions. In contrast, hydrogen bonding interactions are observed between the substrate and Thr379 in the highly conserved "GTGG" motif of JK loop(C), thereby anchoring JK loop(C) in a closed conformation, which secures the appropriate substrate binding mode for catalysis. Site-directed mutagenesis experiments confirm the key role of this residue, highlighting the importance of the JK loop(C) conformation in regulating the enzymatic activity. Furthermore, the existence of the partially and totally open conformations in the substrate-free form suggests a role of JK loop(C) in controlling substrate and product dynamics.

Human embryonic stem cell-derived mesenchymal stromal cells ameliorate collagen-induced arthritis by inducing host-derived indoleamine 2,3 dioxygenase

BackgroundThe immunosuppressive and anti-inflammatory properties of mesenchymal stromal cells (MSC) have prompted their therapeutic application in several autoimmune diseases, including rheumatoid arthritis. Adult MSC are finite and their clinical use is restricted by the need for long-term expansion protocols that can lead to genomic instability. Inhibition of Smad2/3 signaling in human pluripotent stem cells (hPSC) provides an infinite source of MSC that match the phenotype and functional properties of adult MSC. Here, we test the therapeutic potential of hPSC-MSC of embryonic origin (embryonic stem cell-derived mesenchymal stromal cells, hESC-MSC) in the experimental model of collagen-induced arthritis (CIA).MethodsCIA was induced in DBA/1 mice by immunization with type II collagen (CII) in Complete Freund’s Adjuvant (CFA). Mice were treated with either a single dose (106 cells/mouse) of hESC-MSC on the day of immunization (prophylaxis) or with three doses of hESC-MSC every other day starting on the day of arthritis onset (therapy). Arthritis severity was evaluated daily for six weeks and ten days, respectively. Frequency of Treg (FoxP3+), Th1 (IFNγ+) and Th17 (IL17+) CD4+ T cells in inguinal lymph nodes (ILN) was quantified by flow cytometry. Serum levels of anti-CII antibodies were determined by ELISA. Detection of hESC-MSC and quantification of murine and human indoleamine 2,3 dioxygenase (IDO1) expression was performed by quantitative real-time PCR. Statistical differences were analyzed by ANOVA and the Mann-Whitney U test.ResultsAdministration of hESC-MSC to mice with established arthritis reduced disease severity compared to control-treated mice. Analysis of CD4 T cell populations in treated mice showed an increase in FoxP3+ Treg and IFNγ+ Th1 cells but not in Th17 cells in the ILN. Anti-CII antibody levels were not affected by treatment. Migration of hESC-MSC to the ILN in treated mice was associated with the induction of murine IDO1.ConclusionTreatment with hESC-MSC ameliorates CIA by inducing IFNγ+ Th1 cells and IDO1 in the host. Thus, hESC-MSC can provide an infinite cellular source for treatment of rheumatoid arthritis.

NIMG-32MOLECULAR IMAGING AND IMMUNOHISTOCHEMICAL STUDIES OF THE KYNURENINE PATHWAY IN PATIENT-DERIVED XENOGRAFT MODELS OF GLIOBLASTOMA

BACKGROUND: Recently the kynurenine pathway (KP), which is responsible for ∼99% of the catabolism of the amino acid tryptophan (TRP), has been implicated in the pathophysiology of human gliomas. Furthermore, studies indicate that the KP plays a role in local anti-tumor immune suppression both directly and indirectly by depleting TRP and causing T-cell cell cycle arrest. The KP can be studied through the use of a non-invasive molecular imaging tracer, α-[11C]-methyl-L-tryptophan (AMT). AMT-PET can predict survival in GBM patients with recurrent disease (Kamson et al., Neuro-Oncology, 2014). METHODS: In order to study the KP, we generated patient-derived xenograft (PDX) models of five GBMs. One cell line, KCI-GBM-1040, was implanted intracranially and AMT scans were obtained 9 and 14 days post-op. In vivo analysis of the tumors was performed via immunohistochemistry (IHC) and AMT-PET scans. Formalin fixed tissue sections were stained with antibodies specific for human indoleamine 2,3-dioxygenase (IDO) 1, IDO2, tryptophan 2,3-dioxygenase (TDO2), kynureninase (KYNU), kynurenine 3-monooxygenase (KMO), and the large amino acid transporter, LAT1, which is responsible for the uptake of TRP and AMT. RESULTS: Micro-PET imaging showed robust tumoral AMT uptake in PDX mice with prolonged tracer accumulation over a 60-min scan, with average uptake increasing 38% between 10 minutes and the maximum at 50 minutes. This is consistent with AMT trapping seen in human gliomas. Tumor tissues from both the patient and xenografts showed similar immunostaining results for both the TRP-converting and the downstream enzymes of the KP. CONCLUSIONS: These preliminary results indicate that our PDX model is representative of the human disease as it relates to both the in vitro and in vivo study of the KP. These models allow us to study the KP and offer a novel system through which we can develop and test new therapies for GBM patients.

Catalytic activity of human indoleamine 2,3-dioxygenase (hIDO1) at low oxygen

A cytokine-inducible extrahepatic human indoleamine 2,3-dioxygenase (hIDO1) catalyzes the first step of the kynurenine pathway. Immunosuppressive activity of hIDO1 in tumor cells weakens host T-cell immunity, contributing to the progression of cancer. Here we report on enzyme kinetics and catalytic mechanism of hIDO1, studied at varied levels of dioxygen (O2) and l-tryptophan (l-Trp). Using a cytochrome b5-based activating system, we measured the initial rates of O2 decay with a Clark-type oxygen electrode at physiologically-relevant levels of both substrates. Kinetics was also studied in the presence of two substrate analogs: 1-methyl-l-tryptophan and norharmane. Quantitative analysis supports a steady-state rather than a rapid equilibrium kinetic mechanism, where the rates of individual pathways, leading to a ternary complex, are significantly different, and the overall rate of catalysis depends on contributions of both routes. One path, where O2 binds to ferrous hIDO1 first, is faster than the second route, which starts with the binding of l-Trp. However, l-Trp complexation with free ferrous hIDO1 is more rapid than that of O2. As the level of l-Trp increases, the slower route becomes a significant contributor to the overall rate, resulting in observed substrate inhibition.

Human indoleamine 2,3-dioxygenase-2 has substrate specificity and inhibition characteristics distinct from those of indoleamine 2,3-dioxygenase-1

Indoleamine 2,3-dioxygenase-2 (IDO2) is one of three enzymes (alongside tryptophan 2,3-dioxygenase and indoleamine 2,3-dioxygenase (IDO1)) that catalyse dioxygenation of L-tryptophan as the first step in the kynurenine pathway. Despite the reported expression of IDO2 in tumours, some fundamental characteristics of the enzyme, such as substrate specificity and inhibition selectivity, are still to be clearly defined. In this study, we report the kinetic and inhibition characteristics of recombinant human IDO2. Choosing from a series of likely IDO2 substrates, we screened 54 tryptophan derivatives and tryptophan-like molecules, and characterised the 8 with which the enzyme was most active. Specificity of IDO2 for the two isomers of 1-methyltryptophan was also evaluated and the findings compared with those obtained in other studies on IDO2 and IDO1. Interestingly, IDO2 demonstrates behaviour distinct from that of IDO1 in terms of substrate specificity and affinity, such that we have identified tryptophan derivatives that are mutually exclusive as substrates for IDO1 and IDO2. Our results support the idea that the antitumour activity of 1-Me-D-Trp is unlikely to be related with competitive inhibition of IDO2, and also imply that there are subtle differences in active site structure in the two enzymes that may be exploited in the development of specific inhibitors of these enzymes, a route which may prove important in defining their role(s) in cancer.

Substrate Inhibition in Human Indoleamine 2,3-Dioxygenase.

Human indoleamine 2,3-dioxygenase (hIDO) catalyzes the oxidative cleavage of the L-tryptophan (l-Trp) pyrrole ring. Catalysis is inhibited at high substrate concentrations; mechanistic details of this observation are, however, still under debate. Using time-resolved optical spectroscopy, we have analyzed the dynamics of ternary complex formation between hIDO, l-Trp, and a diatomic ligand. The physiological ligand dioxygen (O2) was replaced by carbon monoxide to exclude enzymatic turnover. Quantitative analysis of the kinetics reveals that the ternary complex forms whenever O2 binds first, whereas an l-Trp substrate molecule arriving prior to O2 in the active site causes self-inhibition. Bound l-Trp prevents the ligand from approaching the heme iron and, therefore, impedes formation of the catalytically active ternary complex.

Benzofuranquinones as inhibitors of indoleamine 2,3-dioxygenase (IDO). Synthesis and biological evaluation

A series of benzofuranquinones, analogues of the marine metabolite annulin A, has been synthesized and evaluated as inhibitors of human indoleamine 2,3-dioxygenase (IDO). The synthesis was carried out by copper(II)-mediated reaction of bromobenzoquinones with 1,3-dicarbonyl compounds followed by functional group interconversions. The most potent compounds were 5-methoxy-2-methylbenzofuranquinones containing a CH2OR group at position-3 with IC50 ~ 0.2 mM. The corresponding hydroquinones were inactive. Compounds based on the benzimidazolequinone framework are also active IDO inhibitors. The quinones do not generate significant levels of oxidative stress at concentrations that inhibit IDO.

Synergistic immunomodulatory effects of interferon-gamma and bone marrow mesenchymal stem cells

To investigate mesenchymal stem cells (MSCs) immunosuppressive activity in the presence of interferon-gamma (IFN-γ) to reveal synergistic immunomodulatory effects of IFN-γ and MSCs. ① MSCs were cultured in the presence or absence of IFN-γ（100 ng/ml）, the supernatants were collected for measurements of PGE2、HGF and TGF-β1 by ELISA kits. ② MSCs were cultured in the presence or absence of IFN-γ （100 ng/ml）for 48 h. The cDNA was analysed for the expression of human indoleamine 2, 3-dioxygenase（IDO）mRNA by semiquantitative RT-PCR. ③ Mononuclear cells (MNCs) were extracted from peripheral blood of healthy donors. The T cell proliferation was tested in the co-culture system added with MSCs, recombinant human IFN-γ (100 ng/ml) and anti-IFN-γ mAb (5 μg/ml) by BrdU ELISA kit. ①The immunosuppressive cytokines PGE2、HGF and TGF-β1 were detectable within 24-48 h in the supernatants. Their expressions were significantly up-regulated in the presence of IFN-γ. Concentrations of these cytokines were as of (1715.5±628.6) pg/ml vs (1344.5±709.4) pg/ml (P=0.001);(4031.8±1496.8) pg/ml vs (2452.4±1375.3) pg/ml（P=0.011）;(1753.5±413.8) pg/ml vs (1026.6±450.5) pg/ml（P<0.001）,respectively. ②The expression of IDO mRNA was undetectable when MSCs were cultured alone. In contrast, The IDO mRNA expression was remarkably enhanced in the presence of IFN-γ. ③Bone marrow-derived MSCs remarkably suppressed allogeneic T cell proliferation in vitro. Addition of exogenous IFN-γ had no significant effect on the inhibitory capacity of MSCs, the inhibitory ratios of T cell proliferation were （40.4±10.9）% vs（36.7±7.4）% (P=0.272). By contrast, the inhibitory ratio of T cell proliferation was significantly decreased in the presence of anti-IFN-γ mAb[(40.4±10.9)% vs (23.9±7.6)%,P=0.002]. ①Human MSCs constitutively expressed immunosuppressive concentrations of PGE2, HGF and TGF-β1, and their expressions were significantly up-regulated by IFN-γ. ②IFN-γ-induced expression of IDO on MSCs involved in tryptophan catabolism. ③MSCs notably suppressed allogeneic T cell proliferation in vitro. IFN-γ promoted the immunosuppressive capacity of human MSCs, indicating the synergistic immunomodulatory effect of IFN-γ and MSCs.

How is the distal pocket of a heme protein optimized for binding of tryptophan?

Indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase catalyze the O(2) -dependent oxidation of l-tryptophan to N-formylkynurenine. Both are heme-containing enzymes, with a proximal histidine ligand, as found in the globins and peroxidases. From the structural information available so far, the distal heme pockets of these enzymes can contain a histidine residue (in tryptophan 2,3-dioxygenases), an arginine residue and numerous hydrophobic residues that line the pocket. We have examined the functional role of each of these residues in both human indoleamine 2,3-dioxygenase and human tryptophan 2,3-dioxygenase. We found that the distal histidine does not play an essential catalytic role, although substrate binding can be affected by removing the distal arginine and reducing the hydrophobic nature of the binding pocket. We collate the information obtained in the present study with that reported in the available literature to draw comparisons across the family and to provide a more coherent picture of how the heme pocket is optimized for tryptophan binding.

The mechanism of substrate inhibition in human indoleamine 2,3-dioxygenase.

Indoleamine 2,3-dioxygenase catalyzes the O2-dependent oxidation of l-tryptophan (l-Trp) to N-formylkynurenine (NFK) as part of the kynurenine pathway. Inhibition of enzyme activity at high l-Trp concentrations was first noted more than 30 years ago, but the mechanism of inhibition has not been established. Using a combination of kinetic and reduction potential measurements, we present evidence showing that inhibition of enzyme activity in human indoleamine 2,3-dioxygenase (hIDO) and a number of site-directed variants during turnover with l-tryptophan (l-Trp) can be accounted for by the sequential, ordered binding of O2 and l-Trp. Analysis of the data shows that at low concentrations of l-Trp, O2 binds first followed by the binding of l-Trp; at higher concentrations of l-Trp, the order of binding is reversed. In addition, we show that the heme reduction potential (Em0) has a regulatory role in controlling the overall rate of catalysis (and hence the extent of inhibition) because there is a quantifiable correlation between Em0 (that increases in the presence of l-Trp) and the rate constant for O2 binding. This means that the initial formation of ferric superoxide (Fe3+–O2•–) from Fe2+-O2 becomes thermodynamically less favorable as substrate binds, and we propose that it is the slowing down of this oxidation step at higher concentrations of substrate that is the origin of the inhibition. In contrast, we show that regeneration of the ferrous enzyme (and formation of NFK) in the final step of the mechanism, which formally requires reduction of the heme, is facilitated by the higher reduction potential in the substrate-bound enzyme and the two constants (kcat and Em0) are shown also to be correlated. Thus, the overall catalytic activity is balanced between the equal and opposite dependencies of the initial and final steps of the mechanism on the heme reduction potential. This tuning of the reduction potential provides a simple mechanism for regulation of the reactivity, which may be used more widely across this family of enzymes.

Purification and kinetic characterization of human indoleamine 2,3-dioxygenases 1 and 2 (IDO1 and IDO2) and discovery of selective IDO1 inhibitors

Indoleamine 2,3-dioxygenase (IDO1) catalyzes the first step in tryptophan breakdown along the kynurenine pathway. Therapeutic inhibition of IDO1 is receiving much attention due to its proposed role in the pathogenesis of several diseases including cancer, hypotension and neurodegenerative disorders. A related enzyme, IDO2 has recently been described. We report the first purification and kinetic characterization of human IDO2 using a facile l-tryptophan consumption assay amenable to high throughput screening. We found that the Km of human IDO2 for l-tryptophan is much higher than that of IDO1. We also describe the identification and characterization of a new IDO1 inhibitor compound, Amg-1, by high throughput screening, and compare the inhibition profiles of IDO1 and IDO2 with Amg-1 and previously described compounds. Our data indicate that human IDO1 and IDO2 have different kinetic parameters and different inhibition profiles. Docking of Amg-1 and related analogs to the known structure of IDO1 and to homology-modeled IDO2 suggests possible rationales for the different inhibition profiles of IDO1 and IDO2.

Ferryl Derivatives of Human Indoleamine 2,3-Dioxygenase

The critical role of the ferryl intermediate in catalyzing the oxygen chemistry of monooxygenases, oxidases, or peroxidases has been known for decades. In contrast, its involvement in heme-based dioxygenases, such as human indoleamine 2,3-dioxygenase (hIDO), was not recognized until recently. In this study, H(2)O(2) was used as a surrogate to generate the ferryl intermediate of hIDO. Spectroscopic data demonstrate that the ferryl species is capable of oxidizing azinobis(3-ethylbenzothiazoline-6-sulfonic acid) but not L-Trp. Kinetic studies reveal that the conversion of the ferric enzyme to the ferryl intermediate facilitates the L-Trp binding rate by >400-fold; conversely, L-Trp binding to the enzyme retards the peroxide reaction rate by ∼9-fold, because of the significant elevation of the entropic barrier. The unfavorable entropic factor for the peroxide reaction highlights the scenario that the structure of hIDO is not optimized for utilizing H(2)O(2) as a co-substrate for oxidizing L-Trp. Titration studies show that the ferryl intermediate possesses two substrate-binding sites with a K(d) of 0.3 and 440 μM and that the electronic properties of the ferryl moiety are sensitive to the occupancy of the two substrate-binding sites. The implications of the data are discussed in the context of the structural and functional relationships of the enzyme.

Ligand migration in human indoleamine‐2,3 dioxygenase

Human indoleamine 2,3-dioxygenase (hIDO), a monomeric heme enzyme, catalyzes the oxidative degradation of L-tryptophan (L-Trp) and other indoleamine derivatives. Its activity follows typical Michaelis-Menten behavior only for L-Trp concentrations up to 50 μM; a further increase in the concentration of L-Trp causes a decrease in the activity. This substrate inhibition of hIDO is a result of the binding of a second L-Trp molecule in an inhibitory substrate binding site of the enzyme. The molecular details of the reaction and the inhibition are not yet known. In the following, we summarize the present knowledge about this heme enzyme.

Interplay Between Ligand and Substrate Binding in Human Indoleamine 2,3-Dioxygenase

Human indoleamine 2,3-dioxygenase (hIDO) is a heme-containing enzyme that catalyzes the initial and rate-determining step of L-tryptophan (L-Trp) metabolism via the kynurenine pathway. Earlier kinetic studies showed that the Km of L-Trp (0.015 mM) is ∼27-fold lower than the Kd (0.4 mM) for the ligand-free ferrous enzyme, suggesting that O2 binding proceeds L-Trp binding during the catalytic cycle. With cyanide as a structural probe, we have investigated the thermodynamic and kinetic parameters associated with ligand and substrate binding to hIDO. Kinetic studies demonstrate that pre-binding of L-Trp to the enzyme retards cyanide binding by ∼13-fold, while pre-binding of cyanide to the enzyme facilitates L-Trp binding by ∼22-fold. The data support the view that during the active turnover of the enzyme it is kinetically more favorable to bind O2 prior to L-Trp. Equilibrium titration studies show that the cyanide adduct is capable of binding two L-Trp molecules, with Kd values of 0.018 and 26 mM. The data offer the first direct evidence of the second substrate binding site in hIDO, underlying its substrate-inhibition behavior.

Ligand and Substrate Migration in Human Indoleamine 2,3-Dioxygenase

Human indoleamine 2,3-dioxygenase (hIDO), a monomeric heme enzyme, catalyzes the oxidative degradation of L-tryptophan (L-Trp) and other indoleamine derivatives. Using Fourier transform infrared and optical absorption spectroscopy, we have investigated the interplay between ferrous hIDO, the ligand analog CO, and the physiological substrate L-Trp. These data provide the long-sought evidence for two distinct L-Trp binding sites. Upon photodissociation from the heme iron at T > 200 K, CO escapes into the solvent. Concomitantly, L-Trp exits the active site and, depending on the L-Trp concentration, migrates to a secondary binding site or into the solvent. Although L-Trp is spectroscopically silent at this site, it is still noticeable due to its pronounced effect on the CO association kinetics, which are significantly slower than those of L-Trp-free hIDO. L-Trp returns to its initial site only after CO has rebound to the heme iron.