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Indoleamine-2, 3-dioxygenase (IDO1) and Tryptophan-2, 3-dioxygenase (TDO) catalyze the conversion of L-tryptophan to N-formyl-kynurenine and thus play primary roles in metabolism, inflammation, and tumor immune surveillance. Because their activities depend on their heme contents, which vary in biological settings and go up or down in a dynamic manner, we studied how their heme levels may be impacted by nitric oxide (NO) in mammalian cells. We utilized cells expressing TDO or IDO1 either naturally or via transfection and determined their activities, heme contents, and expression levels as a function of NO exposure. We found NO has a bimodal effect: a narrow range of low NO exposure promoted cells to allocate heme into the heme-free TDO and IDO1 populations and consequently boosted their heme contents and activities 4- to 6-fold, while beyond this range the NO exposure transitioned to have a negative impact on their heme contents and activities. NO did not alter dioxygenase protein expression levels, and its bimodal impact was observed when NO was released by a chemical donor or was generated naturally by immune-stimulated macrophage cells. NO-driven heme allocations to IDO1 and TDO required participation of a GAPDH-heme complex and for IDO1 required chaperone Hsp90 activity. Thus, cells can up- or downregulate their IDO1 and TDO activities through a bimodal control of heme allocation by NO. This mechanism has important biomedical implications and helps explain why the IDO1 and TDO activities in animals go up and down in response to immune stimulation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Violacein is a natural purple pigment of Chromobacterium violaceum with potential medical applications as antimicrobial, antiviral, and anticancer drugs. The initial step of violacein biosynthesis is the oxidative conversion of l-tryptophan into the corresponding α-imine catalyzed by the flavoenzyme l-tryptophan oxidase (VioA). A substrate-related (3-(1H-indol-3-yl)-2-methylpropanoic acid) and a product-related (2-(1H-indol-3-ylmethyl)prop-2-enoic acid) competitive VioA inhibitor was synthesized for subsequent kinetic and x-ray crystallographic investigations. Structures of the binary VioA·FADH2 and of the ternary VioA·FADH2·2-(1H-indol-3-ylmethyl)prop-2-enoic acid complex were resolved. VioA forms a "loosely associated" homodimer as indicated by small-angle x-ray scattering experiments. VioA belongs to the glutathione reductase family 2 of FAD-dependent oxidoreductases according to the structurally conserved cofactor binding domain. The substrate-binding domain of VioA is mainly responsible for the specific recognition of l-tryptophan. Other canonical amino acids were efficiently discriminated with a minor conversion of l-phenylalanine. Furthermore, 7-aza-tryptophan, 1-methyl-tryptophan, 5-methyl-tryptophan, and 5-fluoro-tryptophan were efficient substrates of VioA. The ternary product-related VioA structure indicated involvement of protein domain movement during enzyme catalysis. Extensive structure-based mutagenesis in combination with enzyme kinetics (using l-tryptophan and substrate analogs) identified Arg(64), Lys(269), and Tyr(309) as key catalytic residues of VioA. An increased enzyme activity of protein variant H163A in the presence of l-phenylalanine indicated a functional role of His(163) in substrate binding. The combined structural and mutational analyses lead to the detailed understanding of VioA substrate recognition. Related strategies for the in vivo synthesis of novel violacein derivatives are discussed., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

An intriguing mystery about tryptophan 2,3-dioxygenase is its hydrogen peroxide-triggered enzyme reactivation from the resting ferric oxidation state to the catalytically active ferrous form. In this study, we found that such an odd Fe(III) reduction by an oxidant depends on the presence of L-Trp, which ultimately serves as the reductant for the enzyme. In the peroxide reaction with tryptophan 2,3-dioxygenase, a previously unknown catalase-like activity was detected. A ferryl species (δ = 0.055 mm/s and ΔE(Q) = 1.755 mm/s) and a protein-based free radical (g = 2.0028 and 1.72 millitesla linewidth) were characterized by Mössbauer and EPR spectroscopy, respectively. This is the first compound ES-type of ferryl intermediate from a heme-based dioxygenase characterized by EPR and Mössbauer spectroscopy. Density functional theory calculations revealed the contribution of secondary ligand sphere to the spectroscopic properties of the ferryl species. In the presence of L-Trp, the reactivation was demonstrated by enzyme assays and by various spectroscopic techniques. A Trp-Trp dimer and a monooxygenated L-Trp were both observed as the enzyme reactivation by-products by mass spectrometry. Together, these results lead to the unraveling of an over 60-year old mystery of peroxide reactivation mechanism. These results may shed light on how a metalloenzyme maintains its catalytic activity in an oxidizing environment.

Pyrrolnitrin (3-chloro-4-(2'-nitro-3'-chlorophenyl)pyrrole) is a broad-spectrum antifungal compound isolated from Pseudomonas pyrrocinia. Four enzymes (PrnA, PrnB, PrnC, and PrnD) are required for pyrrolnitrin biosynthesis from tryptophan. PrnB rearranges the indole ring of 7-Cl-l-tryptophan and eliminates the carboxylate group. PrnB shows robust activity in vivo, but in vitro activity for PrnB under defined conditions remains undetected. The structure of PrnB establishes that the enzyme belongs to the heme b-dependent indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) family. We report the cyanide complex of PrnB and two ternary complexes with both l-tryptophan or 7-Cl-l-tryptophan and cyanide. The latter two complexes are essentially identical and mimic the likely catalytic ternary complex that occurs during turnover. In the cyanide ternary complexes, a loop previously disordered becomes ordered, contributing to the binding of substrates. The conformations of the bound tryptophan substrates are changed from that seen previously in the binary complexes. In l-tryptophan ternary complex, the indole ring now adopts the same orientation as seen in the PrnB binary complexes with other tryptophan substrates. The amide and carboxylate group of the substrate are orientated in a new conformation. Tyr(321) and Ser(332) play a key role in binding these groups. The structures suggest that catalysis requires an l-configured substrate. Isothermal titration calorimetry data suggest d-tryptophan does not bind after cyanide (or oxygen) coordinates with the distal (or sixth) site of heme. This is the first ternary complex with a tryptophan substrate of a member of the tryptophan dioxygenase superfamily and has mechanistic implications.

Indoleamine 2,3-dioxygenase is a heme enzyme that catalyzes the oxidative degradation of L-Trp and other indoleamines. We have used resonance Raman spectroscopy to characterize the heme environment of purified recombinant human indoleamine 2,3-dioxygenase (hIDO). In the absence of L-Trp, the spectrum of the Fe(3+) form displayed six-coordinate, mixed high and low spin character. Addition of L-Trp triggered a transition to predominantly low spin with two Fe-OH(-) stretching modes identified at 546 and 496 cm(-1), suggesting H-bonding between the NH group of the pyrrole ring of L-Trp and heme-bound OH(-). The distal pocket of Fe(3+) hIDO was explored further by an exogenous heme ligand, CN(-); again, binding of L-Trp introduced strong H-bonding and/or steric interactions to the heme-bound CN(-). On the other hand, the spectrum of Fe(2+) hIDO revealed a five-coordinate and high spin heme with or without L-Trp bound. The proximal Fe-His stretching mode, identified at 236 cm(-1), did not shift upon L-Trp addition, indicating that the proximal Fe-His bond strength is not affected by binding of the substrate. The high Fe-His stretching frequency suggests that Fe(2+) hIDO has a strong "peroxidase-like" Fe-His bond. Using CO as a structural probe for the distal environment of Fe(2+) hIDO revealed that binding of L-Trp in the distal pocket converted IDO to a peroxidase-like enzyme. Binding of L-Trp also caused conformational changes to the heme vinyl groups, which were independent of changes of the spin and coordination state of the heme iron. Together these data indicate that the strong proximal Fe-His bond and the strong H-bonding and/or steric interactions between l-Trp and dioxygen in the distal pocket are likely crucial for the enzymatic activity of hIDO.

Tryptophan 2,3-dioxygenase (EC 1.13.1.12) is a hemoprotein which catalyzes the first step in the oxidative degradation of tryptophan. The reaction is believed to proceed by addition of O2 across the 2,3-bond of the indole ring, followed by decomposition of the resultant dioxetane to give N-formylkynurenine. A primary D2O isotope effect of 4.4 on Vmax/Km was observed at the pH optimum, pH 7.0. This implies that abstraction of the indole proton is at least partially rate-determining. An inverse secondary isotope effect of 0.96 was observed for L-[2-3H]tryptophan at this pH. The secondary isotope effect signals the formation of the C-O bond at C-2. As the rate of proton abstraction increased with increasing pH, the D2O isotope effect decreased to 1.2 at pH 8.5 and the secondary isotope effect increased to 0.92. The rate-determining steps therefore change with increasing pH, and bond formation at C-2 becomes more rate-limiting. The secondary isotope effect did not change significantly with varying O2 concentration so that substrate binding is primarily ordered with O2 binding first. The specificity of the enzyme towards substituted tryptophans shows that substitution of the phenyl ring of the indole is sterically unfavorable. Steric hindrance is highest at the 4- and 7-positions, while the 5- and 6-positions are less sensitive. 6-Fluoro-L-tryptophan was more reactive than tryptophan, and the increased reactivity can be explained by an electronic effect that enhances of the rate of C-O bond formation at C-2.

The basal activity of tryptophan 2,3-dioxygenase (EC-1.13.11.11) in primary cultured rat hepatocytes decreased during culture, but addition of either tryptophan (2.5 x 10(-3) M) or dexamethasone (1 x 10(-6) M) could prevent the decrease. Addition of both compounds caused severalfold induction of activity. Glucagon (1 x 10(-8) M) alone did not induce the activity, but its inductive effect in combination with tryptophan was similar to that of tryptophan plus dexamethasone. The effect of glucagon was additive with those of tryptophan and dexamethasone and hence the highest induction (7-fold) was achieved by addition of all three inducers. Glucagon could be replaced by dibutyryl cyclic AMP (1 x 10(-5) M). Insulin (1 x 10(-8) M) inhibited the inductions by glucagon and dexamethasone, but not that by tryptophan. Cycloheximide inhibited the inductions by all three inducers, but actinomycin D inhibited only the induction by dexamethasone. These results suggest that the three compounds have different mechanisms of induction of tryptophan oxygenase activity: tryptophan prevents enzyme inactivation, dexamethasone may stimulate enzyme synthesis at the level of transcription, and glucagon may enhance the synthesis at the translational level.

In primary cultures of rat hepatocytes, addition of dexamethasone (10 microM) plus glucagon (0.5 microM) caused several-fold increases in the activities of serine dehydratase (EC 4.2.1.13), tryptophan oxygenase (EC 1.13.11.11), and tyrosine aminotransferase (EC 2.6.1.5) in 24 h. These inductions were inhibited by insulin. Addition of epinephrine or phenylephrine at 10 microM blocked these inductions. This suppressive effect of adrenergic compounds was completely abolished by the alpha-adrenergic antagonist phenoxybenzamine at 10 microM. Immunochemical analysis with antiserum to serine dehydratase showed that the changes in enzyme activity were due to changes in the amount of enzyme. Epinephrine was effective even when glucagon was replaced by dibutyryl cAMP (50 microM), indicating that alpha-adrenergic suppression of enzyme inductions was mediated by a cAMP-independent mechanism. Furthermore, the findings that prazosin antagonized this epinephrine effect, but yohimbine did not, indicate that the alpha 1- but not the alpha 2-receptor is involved in this inhibition. However, the alpha-adrenergic effect was different from that of insulin in that, unlike the latter, the inductions of tryptophan oxygenase and tyrosine amino-transferase by dexamethasone alone were not inhibited. The alpha-adrenergic action apparently counteracts the action of glucagon and cAMP. For determination of the beta-adrenergic effect of catecholamines on the inductions of enzymes, beta-adrenergic compounds were tested without glucagon. Isoproterenol or epinephrine plus phenoxybenzamine induced tryptophan oxygenase and tyrosine aminotransferase. Induction of serine dehydratase was shown by isoproterenol only in the presence of 1-methyl-3-isobutylxanthine, an inhibitor of phosphodiesterase. These results indicate that catecholamines play dual roles in regulation of the amount of enzyme through their alpha 1- and beta-adrenergic actions.

Tryptophan oxygenase activity follows a characteristic developmental pattern in the young rat, being absent until about 2 weeks of life, then attaining adult levels by the 3rd week. We have investigated the factors which control this process and determined that the increase in enzymatic activity results from increased glucocorticoid release between the 14th and 21st days. This conclusion was based on our observation that adrenalectomy completely arrests the development of the enzymatic activity. Twenty-one-day-old animals which were adrenalectomized on the 10th day, and thus possess no demonstrable enzymatic activity, could respond to cortisol treatment with an elevation of tryptophan oxygenase activity. Administration of either cycloheximide or actinomycin D completely inhibited this response, suggesting that both protein synthesis and RNA synthesis were requisite for the development of the enzyme. Immunochemical studies revealed that the enzyme found in the young animal was identical with that extracted from the adult rat; furthermore, the increase in activity observed in the developing rat is the result of increased enzyme protein levels. Direct measurement of the synthetic and degradative rates showed the accumulation of enzyme protein to depend upon increased synthetic activity, and not upon decreased enzyme degradation.

Exogenously administered heme is incorporated into rat hepatic cytochrome P-450 in vivo (Correia, M. A., Farrell, G. C. Schmid, R. S., Ortiz de Montellano, P. R., Yost, G. S., and Mico, B. A. (1979) J. Biol. Chem. 254, 15-17). This was demonstrated in allylisopropylacetamide (AIA)-treated rats by the formation of a radioactive adduct derived from the porphyrin of the administered [3H]heme and AIA. Formation of such adducts requires catalytic participation of cytochrome P-450 in oxidative metabolism of AIA to an active species which subsequently alkylates the prosthetic heme moiety of the cytochrome. These results suggested that the exogenous heme had been incorporated prosthetically into cytochrome P-450 prior to generation of the adduct. However, the possibility remained that a minute portion of the inactivating AIA-species escaped the catalytic site of the generating hemoprotein and alkylated the nonprosthetically bound isotopic heme. To examine this critical possibility, we have employed a chemical derivative of heme which binds to the microsomal membrane. Although this heme derivative is a structurally suitable target for attack by the inactivating drug species, we found that it was unsuitable for incorporation into the prosthetic site of cytochrome P-450. The findings of this study provide irrefutable evidence that the label recovered in drug-porphyrin adducts is derived exclusively from radioactive heme incorporated prosthetically into cytochrome P-450. Drug-porphyrin adducts can therefore be used as reliable probes to follow the transfer of heme from the hepatic "free" heme pool into cytochrome P-450.

We investigated how changes in tryptophan pyrrolase activity and tryptophan loads affect the breakdown of tryptophan was estimated by injecting rats with [ring-2-14-C]tryptophan and measuring respiratory 14-CO2. We concluded, contrary to previous reports, that induction of tryptophan pyrrolase definitely will increase the rate of tryptophan breakdown. Tryptophan loads also increase tryptophan breakdown even in circumstances where there is no increase in tryptophan pyrrolase activity, presumably by increasing the saturation of the enzyme. After a tryptophan load (50 mg per kg) the increase in liver tryptophan concentration lasts only 30 min. The rapid return of liver tryptophan to normal may be due partly to the high turnover rate of liver tryptophan. We estimate that tryptophan pyrrolase degrades tryptophan in vivo at a rate that is equivalent to the whole liver tryptophan concentration in 7.5 min or less.

Carbonmonoxy indoleamine 2,3-dioxygenase from rabbit small intestine exhibited two CO stretch bands at 1953 and 1933 cm-1 with half-band widths (delta v 1/2) of both approximately 15 cm-1. Upon addition of an excess amount of L-tryptophan, the substrate, the spectrum changed into that with an intense single band at 1902 cm-1 with the delta v 1/2 of 15 cm-1. Carbonmonoxy L-tryptophan 2,3-dioxygenase of Pseudomonas acidovorans in the absence of L-tryptophan showed a fused CO stretch band which consists of two components at 1965 and 1958 cm-1 (delta v 1/2 for the fused band; 25 cm-1), which was converted into a sharp single band at 1968 cm-1 (delta v 1/2; 10 cm-1) upon addition of excess L-tryptophan. On the other hand, CO complex of rat liver L-tryptophan 2,3-dioxygenase in the absence of L-tryptophan gave a spectrum with a poorly defined peak around 1961 cm-1. By the addition of L-tryptophan, the spectrum changed into that with two distinct bands at 1972 and 1920 cm-1 (delta v 1/2; 6 and 13 cm-1, respectively). These spectra were insensitive to pH in a range where the enzymes were not denatured (neutral to near pH 9). The infrared spectra of the carbonmonoxy enzymes were also affected by the addition of certain effectors such as skatole and alpha-methyl-DL-tryptophan, which facilitate the binding of L-tryptophan to the catalytic site of intestinal and Pseudomonas enzymes, respectively. However, the changes were of different types from those by the saturating amount of L-tryptophan. Possible mechanisms for these phenomena are discussed in relation to the structure of the heme-CO complex in these heme-containing dioxygenases.

Allosteric interactions in the cupro-heme enzyme tryptophan oxygenase (EC 1.13.11.11) of Pseudomonas acidovorans are shown to be pH-dependent. Increasing the assay pH from 6.0 to 8.0 progressively desensitizes the enzyme from both homotropic and heterotropic ligand interactions. This pH-dependent reversible transition has a pK of 6.2. Hill coefficients for the substrate L-tryptophan of 2.0 and 1.4 were measured at pH 6.0 and pH 7.0, respectively. In attempting to identify the enzymatic residue (or residues) responsible for these pH-dependent effects, the enzyme was observed to be irreversibly inactivated by photoinduced oxidation in the presence of the sensitizer, methylene blue. The photoinactivated enzyme showed a loss of one-half its Soret (405 nm) absorption which accompanied the loss of one-half its heme and histidine contents. This first order photoinduced inactivation was pH-dependent and corresponded to a requirement for a protonated species with a pK of 6.2. These results suggest that histidine residues may be involved in the catalytic function and in mediating cooperative interactions of tryptophan oxygenase. Absolute and difference sedimentation velocity analyses indicate that the molecule undergoes a conformational transition when the pH is decreased from pH 8.0 to pH 6.0. This conformational alteration, measured as a 3.9% increase in S20, w can be regarded as an equivalent decrease in the frictional coefficient. If, a more or less spherical shape to the molecule is assumed, then, the 3.9% decrease in the frictional coefficient between pH 8.0 and 6.0 corresponds to a 12% decrease in apparent hydrodynamic volume of the enzyme. Thus, protonation of an enzymatic moiety, possibly histidine, determines both the conformational and functional interactions between enzymatic sites.

Induction by interferon-gamma of indoleamine 2,3-dioxygenase (a tryptophan degradation enzyme) was examined with 11 human cell lines. The enzyme induction was demonstrated in 7 of the 11 cell lines. The induced enzyme in each of the 7 cell lines was identical to the enzyme purified from human placenta, as evidenced by immunoblot analysis with a monoclonal antibody specific to the placental one. The extent of the induction varied largely with the cell line; a relatively high induction was observed with HEL (lung fibroblasts), NY (osteosarcoma), and A-431 (epidermoid carcinoma). The enzyme induction was dependent on the concentration of interferon-gamma and occurred 12-18 h after addition of interferon-gamma to the cultures. Interferon-alpha or -beta was completely ineffective in this induction. Interferon-gamma inhibited the growth of the 7 cell lines observed with the enzyme induction, and this growth inhibition was accompanied with a complete deletion of tryptophan (less than 1 microM) in the culture medium by the induction of the enzyme. For two of these cell lines, the inhibition was partially reversed by an addition of exogenous tryptophan to the medium not to be depleted. These findings indicated that the growth inhibition by interferon-gamma was in part explained by the tryptophan depletion in the medium caused by the enzyme induction.

The oxygenated complexes of the two catalytically active forms of pseudomonad and rat liver L-tryptophan-2,3-dioxygenase (EC 1.13.11.11) have been studied. As was previously reported (ISHIMURA, Y., NORZAKI, M., HAYAISHI, O., TAMURA, M., AND YAMAZAK-I I. (1970) J. Biol. Chem. 245, 3593-3602), we observe that the fully reduced form of pseudomonad tryptophan oxygenase during steady state catalysis exists predominantly as the L-tryptophan ferroheme-O2 enzyme complex (lambdamax = 415 nm, 540 nm, 570 nm). However, during steady state catalysis by a half-reduced form of both the pseudomonad and hepatic enzymes, the predominant species present manifest absorption spectra indicative of ternary complexes in which all the heme exists as ferriheme (Soret, 407 nm), there being no trace of a ferroheme-O2 complex. Carbon monoxide is a competitive inhibitor with respect to molecular oxygen of catalysis by either the half-reduced or fully reduced forms of pseudomonad tryptophan oxygenase. During steady state catalysis in the presence of CO, the fully reduced form of the enzyme exists as a mixture of the oxyferroheme (Soret = 415 nm) and carboxyferroheme (Soret = 421 nm) enzyme complexes. However, if the same experiment is repeated with the half-reduced form of the pseudomonad enzyme, all of the enzyme is in the ferriheme state, even though CO is inhibiting this form of the enzyme to the same degree as it does the fully reduced form. We conclude that for the half-reduced form of pseudomonad tryptophan oxygenase the substrate, O2, and the inhibitor, CO, are not binding to the heme moieties, but are bound elsewhere, presumably to the Cu(I) moieties. Examination of the kinetic mechanisms of the half-reduced and fully reduced forms of pseudomonad tryptophan oxygenase using the inhibitors carbon monoxide and 5-fluorotryptophan confirmed that the fully reduced enzyme binds L-tryptophan before O2 (FORMAN, H., AND FEIGELSON, P. (1971) Biochemistry 10, 760-763) and that for the half-reduced enzyme O2 binds first. In the presence of 5-fluorotryptophan a relatively stable oxyferroheme enzyme complex was generated with the fully reduced form of pseudomonad tryptophan oxygenase. Thus, saturation of the catalytic site alone either with the substrate, L-tryptophan, or the competitive inhibitor, 5-fluorotryptophan, enhances binding of O2 to the ferroheme moieties of the enzyme. The resistance of this complex to photolysis indicates that the bound molecular oxygen is predominantly present as superoxide, O2-minus.