Potent Activation of Indoleamine 2,3-Dioxygenase by Polysulfides

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Potent Activation of Indoleamine 2,3-Dioxygenase by Polysulfides Micah T. Nelp, Vincent Zheng, Katherine M. Davis, Katherine Stiefel, and John T. Groves J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b07338 • Publication Date (Web): 22 Aug 2019 Downloaded from pubs.acs.org on August 22, 2019

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Potent Activation of Indoleamine 2,3-Dioxygenase by Polysulfides Micah T. Nelp, Vincent Zheng, Katherine M. Davis, Katherine J. E. Stiefel and John T. Groves* Department of Chemistry, Princeton University, Princeton, New Jersey 08544, United States ABSTRACT: Indoleamine 2,3-dioxygenase (IDO1) is a heme enzyme that catalyzes the oxygenation of the indole ring of tryptophan to afford Nformylkynurenine. This activity significantly suppresses the immune response, mediating inflammation and auto-immune reactions. These consequential effects are regulated through redox changes in the heme cofactor of IDO1, which autoxidizes to the inactive ferric state during turnover. This change in redox status increases the lability of the heme cofactor leading to further suppression of activity. The cell can thus regulate IDO1 activity through the supply of heme and reducing agents. We show here that polysulfides bind to inactive ferric IDO1 and reduce it to the oxygenbinding ferrous state, thus activating IDO1 to maximal turnover even at low, physiologically significant concentrations. The on-rate for hydrogen disulfide binding to ferric IDO1 was found to be >106 M-1 s-1 at pH 7 using stopped flow-spectrometry. K-edge XANES and EPR spectroscopy indicated initial formation of a low-spin ferric sulfur-bound species followed by reduction to the ferrous state. The µM affinity of polysulfides for IDO1 implicates these polysulfides as important signaling factors in immune regulation through the kynurenine pathway. Tryptophan significantly enhanced the relatively lower-affinity binding of hydrogen sulfide to IDO1, inspiring the use of the small molecule 3-mercaptoindole (3MI), which selectively binds to and activates ferric IDO1. 3MI sustains turnover by catalytically transferring reducing equivalents from glutathione to IDO1, representing a novel strategy of upregulating innate immunosuppression for treatment of autoimmune disorders. Reactive sulfur species are thus likely unrecognized immune-mediators with potential as therapeutic agents through these interactions with IDO1.

Introduction Indoleamine 2,3 dioxygenase (IDO1) is a heme enzyme that catalyzes the dioxygenation of the indole ring of tryptophan and similar substrates such as melatonin, serotonin, and tryptamine.1-2 IDO1, along with the paralogous indoleamine 2,3 dioxygenase-2 and tryptophan dioxygenase, oxidize tryptophan to N-formylkynurenine in the first committed step of the kynurenine pathway that leads to the synthesis of the nicotinamide adenine dinucleotide cofactor.3-4 The vast majority of tryptophan, an essential and energetically expensive amino acid, is catabolized through this pathway.4-6 The mechanism of IDO1 is considered to entail oxygen-binding to the ferrous enzyme to form the ferric-superoxide complex. This species is proposed to directly engage with the indole ring of its substrates via an alkylperoxo-iron(III) intermediate to form an indole epoxide and ferryl intermediates, both of which have been obFigure 1. The oxygenation of tryptophan by IDO1 suppresses the imserved.7-8 Recombination of these fragments leads to oxidative scismune response and is itself regulated by redox control. During turnover, sion of the indole ring and regeneration of the enzyme in the active IDO1 is prone to loss of superoxide resulting in the inactive ferric state ferrous state (Figure 1).9-10 that is unable to bind to substrate oxygen until it is reduced to the ferrous state. Ferric IDO1 is prone to heme loss. The oxidation of tryptophan through IDO1 activity is central to immune regulation, providing a necessary brake to prevent autoimthat IDO1 is essential for fetal tissue to survive the maternal immune munity and damaging inflammation.11 IDO1 is expressed in many response.13 The remarkable ability of IDO1 to induce immune tolertissues, but it is highly upregulated in response to infection signaled ance is also utilized by cancer cells, many of which demonstrate high by the cytokine interferon-γ and lipopolysaccharides.11 By depleting expression of IDO1 that correlates with poor prognoses.14-15 Accordtryptophan from the local tissue environment, IDO1 stalls the proingly, intense efforts been made to find inhibitors of IDO1 to restore liferation of invading pathogens as well as immune cells. The resultnormal immune clearance of cancer cells.16 Alternatively, the downant production of kynurenine metabolites causes similar effects by regulation of IDO1 activity potentiates many autoimmune disortriggering cell cycle arrest and promoting the generation of immune ders, including rheumatoid arthritis, type I diabetes, multiple sclerosuppressing T-regulatory cells.12 sis, and inflammation leading to cardiovascular disease.17-20 Immunosuppression by IDO1 was discovered when it was found ACS Paragon Plus Environment

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Activation of IDO1 is thus likely to have similarly far-reaching effects in health as its inhibition, but this aspect of IDO1 regulation has only just begun to be explored.17 No specific activators of tryptophan oxidation by IDO1 have been reported to date. The cellular regulation of IDO1 is unsurprisingly intricate. At the post-translational level alone, IDO1 activity is affected by phosphorylation21, ubiquitination22, nitration23, nitrosylation24, allosteric binding of small molecules25, and the dynamic binding of its heme cofactor.26-28 The redox state of the enzyme provides yet another important lever of control. To bind molecular oxygen and activate tryptophan, IDO1 must be in the reduced ferrous state, where it returns in its catalytic cycle without the need for additional reducing equivalents. And yet, if no reductant is supplied during multiple turnovers, IDO1 undergoes abortive autoxidation with loss of superoxide to produce the inactive ferric enzyme.29 The ferric IDO1 produced in this manner persists even in the reducing environment of cells,30 suggesting that self-limiting autoxidation is an adaption to prevent excessive activity. Further highlighting the importance of redox regulation in tryptophan metabolism, inactive ferric IDO1 is also prone to long-term inactivation through heme-loss, which likely accounts for the majority of cellular IDO1 found in the apo-form.26, 31 Regeneration and maintenance of active, ferrous IDO1 in cells

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has been attributed to cytochrome b5.30, 32-33 We sought to explore and understand what other cellular reductants might affect the IDO1 redox switch and, by implication, the immune response. Our initial efforts were motivated by recent work describing the hydrogen sulfide-mediated reduction of myoglobin, hemoglobin, heme oxygenase, and porphyrin complexes.34-40 Hydrogen sulfide binds to the ferric heme of these proteins to form a hydrosulfido complex that can then lead to reduction to the ferrous heme and production of sulfhydryl radical,41 a paradigm matching the requirements for IDO1 activation in cells. The final oxidized products of these reactions, polysulfides, have also gained attention with the discovery of their high, micromolar concentrations within cells and their remarkably versatile reactivity, including ferric heme reduction in cytochrome c.42-46 Here, we show that hydrogen sulfide and, to a much greater extent, polysulfides bind to IDO1 and reduce it to the active ferrous state, linking the immune response in a novel way to sulfide signaling. We further show that a small-molecule thiol, 3-mercaptoindole, replicates these effects, suggesting a new class of selective IDO1reducing agonists. Results Interaction of Polysulfides and IDO1. Polysulfides (HSnH) were found to bind rapidly to heterologously expressed human

Figure 2. Sodium disulfide (Na2S2) binds, reduces and activates IDO1. (A) Stopped-flow UV-vis spectrometry of the binding of 5µM Na2S2 and 2.6 µM IDO1 at pH 7.0. (B) Stopped-flow UV-vis spectrometry of IDO1 (150 nM) and Na2S2 used to obtain an upper limit for the Kd. (C) Second-order rate constant for Na2S2 binding to IDO1 (2 µM) determined by stopped flow spectrometry. (D) Kinetic parameters for Na2S2 binding to IDO1 (500 nM) fit to a Michaelis-Menten expression with L-tryptophan (50 µM) as substrate.

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IDO1, as evidenced by significant changes in its UV-vis spectrum upon rapid mixing at pH 7.0 using stopped-flow spectrometry. The predominate polysulfide species at this pH are HSn– (see experimental). Hydrogen disulfide, trisulfide, and tetrasulfide all produced similar spectra with the Soret λmax , shifting from 404 nm in the ferric form to 426 nm with a smaller peak at 370 nm in the disulfide adduct. The q-bands of IDO1 also shifted upon polysulfide binding from a broad peak centered at 500 nm to two peaks at 546 nm and a shoulder centered around 578 nm (Figures 2A and S1A). Reduction of the polysulfido-IDO1 complex to ferrous IDO1 was demonstrated using UV-vis spectrometry. Due to the potentially interfering and fast reaction of ferrous IDO1 with molecular oxygen (5.3 × 105 M−1 s−1),47 the reaction buffer was placed under vacuum just prior to use, and the headspace was flushed with nitrogen gas. The reduction of IDO1 by hydrogen disulfide was clearly signaled by the shift of the ferric heme-polysulfido UV-vis peak at 546 nm and shoulder centered around 578 to a single peak at 550 nm that is characteristic of ferrous IDO1 (Figure S2A).48 Complete reduction occurred within 400 s with an estimated rate of reduction of 8.5 ± 0.4 x 10-3 s-1. The Kd for polysulfides and IDO1 was found to be less than 500 nM, consistent with complete conversion to the polysulfido complex with only 5 µM polysulfide (Figure 2A). A more accurate estimate was obtained using stopped-flow spectrometry to observe the

bound species within 1 s after mixing. This technique avoids interfering secondary reactions such as reduction and subsequent reaction of the ferrous IDO1 with molecular oxygen. Using 150 nM IDO1, it was found that 0.5 µM hydrogen disulfide was sufficient to completely bind to IDO1 giving a maximal Kd of 50 nM (Figure 2B). The on-rate for hydrogen disulfide with IDO1 was found to be 2.6 x 106 M-1 s-1 following the reaction by the increase in absorbance at the λmax of the disulfide-bound species at 425 nm (Figure 2C). Using the estimated Kd of 50 nM, this provides a lower limit for the disulfide off-rate of ~ 0.1 s-1, similar to the rate of reduction. Hydrogen disulfide was mixed with IDO1 in the presence of substrates tryptophan and molecular oxygen (at ambient concentration in solution, pH 7.0) to see if the resultant reduction could activate the enzyme for turnover. With these aerobic conditions, 50 µM Ltryptophan, and various concentrations of hydrogen disulfide, turnover was observed (Figure 2D). The turnover rate was plotted against polysulfide concentration and fit using a Michaelis-Menten expression to give a maximal turnover rate of 1.4 ± 0.1 s-1, comparable to the commonly used ascorbate/methylene blue assay (1-2 s1 26, 49 ) and that of cytochrome b5 (1.7-10 s-1 ).33, 50 Polysulfides form an equilibrium mixture of chain lengths in neutral aqueous solution,

Figure 3. Interaction of hydrogen sulfide with IDO1. (A) UV-vis spectrometry of IDO1 (5 µM) titrated with sodium sulfide (0-12.8 mM at pH 7.0 with concentrations matching data points in B). (B) The binding constant of sodium sulfide and IDO1 derived from (A). (C) Second-order rate constant of sodium sulfide binding to IDO1 (2 µM) determined by stopped flow spectrometry. (D) The activation parameters of sulfide and IDO1 (200 nM) fit to a Michaelis-Menten expression and a constant of inhibition with L-tryptophan (50 µM) as substrate.


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but the Km for reductive activation was found to be different depending on the proffered polysulfide: 8 ± 1 µM for Na2S2 (Figure 2D), 3 ± 0.7 µM for Na2S3, and 11 ± 2 µM for Na2S4 (Figure S1B). Interaction of Hydrogen Sulfide with IDO1. Hydrogen sulfide was found to bind to IDO1 producing a UV-vis spectrum quite similar to that of the polysulfide-IDO1 complexes with the Soret λmax of 404 nm of ferric IDO1 shifting to a Soret at λmax 420 nm and a smaller peak at 355 nm. These are slightly blue-shifted from the comparable peaks in the polysulfido-IDO1 complex. The q-bands are also similar though more pronounced relative to the polysulfido complex (Figure 3A). The pKa of hydrogen sulfide is 6.8,52 and so the protonated (H2S) or deprotonated (HS–) species responsible for binding was tested by varying the pH from 5.1 to 8.2. The rate of binding was significantly increased at higher pH suggesting the singly deprotonated form (HS-) is the species that binds to the ferric heme in IDO1 (Figure S3). The Kd for this hydrosulfide-IDO1 complex was determined to be 0.34 ± 0.02 mM by titrating sodium sulfide with IDO1 and then using linear regression to globally fit each resultant spectrum to a series of combinations of bound and unbound spectra (Figure 3B). Stopped-flow spectrometry enabled a second means of determining the on- and off-rates by treating the data as an approach to equilibrium.53 Varying the concentration of hydrogen sulfide under pseudo-first-order binding conditions produced rates that fit well to a line, the slope of which corresponds to the on-rate, 1.4 x 105 M-1 s1 , and the y-intercept to the off-rate, 55 s-1 (Figure 3C). The Kd from these kinetic parameters (off-rate/on-rate) is 0.39 mM. The ability of hydrogen sulfide to reduce IDO1 was examined with UV-vis spectrometry as with polysulfides, adding a sodium sulfide solution to a final concentration of 10 mM (Figure S2B). The IDO1-hydrogen sulfide complex formed in under 10 s. Its spectrum showed a gradual change over 200 s with the Soret shifting from 421 nm to 430 nm as well as a change in the q-band peaks from two at 545 and 573 nm shifting to a single large peak centered at 550 nm, all consistent with ferrous IDO1 accumulating and becoming the major species.48 Hydrogen sulfide is thus capable of binding to IDO1 and reducing it to the ferrous, active form. Complete reduction occurred in about 300 s with an estimated rate of reduction of 2.3 ± 0.2 x 10-2 s-1, somewhat faster than with hydrogen disulfide (8.5 ± 0.4 x 10-3 s-1), though in this comparison the hydrogen sulfide was significantly more concentrated (5 mM compared to 100 µM disulfide), which could presumably lessen the effects of side reactivity such as with residual oxygen. The activating ability of sulfide with IDO1 showed an initial lag phase, a characteristic shared with hydrogen sulfide oxidation catalyzed by myoglobin35 (Figures S4). The rates reported here were fit to the linear portions of these activity profiles. The ability of hydrogen sulfide to activate IDO1 was modeled using a Michaelis-Menten expression as was done with hydrogen disulfide. The IDO1 turnover rate with sulfide was 1.2 ± 0.1 s-1, and the Km for IDO1 activation was 0.5 ± 0.1 mM. At higher concentrations of hydrogen sulfide, the turnover rate began to decrease, and so this phase was fit using an expression for substrate inhibition resulting in a Ki of 6.0 ± 2 x 10-2 M (Figure 3D). The product of hydrogen sulfide oxidation by other heme proteins has been reported to be polysulfides and thiosulphate.34-36 To test for the production polysulfides from hydrogen sulfide and IDO1, sulfane sulfur probe 4 or SSP4,54 a fluorescent probe that is specific for detection of polysulfides, was added to mixtures of IDO1

100

Percent IDO1 Bound to Sulfide

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80

60 [L-Trp] (µM) 0 50 100 200 300 400 600 800

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0.2

0.4 0.6 [Na S] (mM)

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Figure 4. Tryptophan and Hydrogen Sulfide Bind Cooperatively to IDO1. Stopped flow UV-vis spectrometry was used to determine the extent of sodium sulfide binding to IDO1 with various amounts of L-tryptophan (0-800 µM) at pH 7.0.

and sulfide that had been incubated for 1 h. Fluorescence spectrometry showed fluorescein production, indicative of the presence of polysulfides, only when IDO1 and sulfide were combined but not with either alone. Thus, IDO1 is capable of producing polysulfides from hydrogen sulfide (Figure S5). Tryptophan and Hydrogen Sulfide Bind Cooperatively with IDO1. The binding of hydrogen sulfide to ferric IDO1 showed a Kd (0.34 ± 0.02 mM) out of range of the physiological concentrations of hydrogen sulfide, with some reports suggesting only nM concentrations.55 Sulfide binding was significantly enhanced in the presence of L-tryptophan, decreasing the Kd from ~ 0.3 mM to 0.05 mM at the higher end of tryptophan cellular availability (~ 200 µM).56 The sulfide Kd is decreased even further to 12 µM when tryptophan concentration is raised to 800 µM (Figure 4). This aspect was tested using stopped-flow spectrometry to prevent interference from resultant turnover. Interestingly, the spectrum of hydrosulfido-IDO1 is not significantly altered by high concentrations of L-tryptophan despite its stabilizing effect (Figure S6) - a slight blue-shifting in the Soret from λmax at 424 nm to 422 nm and a slight red shifting of the q-band peaks are all that are evident, and even these small changes may be attributable to contaminating ternary complex with molecular oxygen that could be starting to form.29 3-Mercaptoindole Activates IDO1 Selectively. The interaction of tryptophan in stabilizing hydrosulfido-IDO1 suggested that a small molecule that combines features of both tryptophan and hydrogen sulfide might be able to take advantage of both binding capabilities to produce an overall tighter binding affinity with IDO1. 3Mercaptoindole (3MI) was seen to bind to IDO1 with a fast on-rate of 1.7 x 106 M-1 s-1 under pseudo first-order conditions (Figure 5B), producing a UV-vis spectrum similar to those observed with hydrogen sulfide and polysulfides: a Soret at 424 nm, a lower peak at 321 nm, and broad q-bands at 537 nm and a shoulder at about 575 nm (Figure 5A). The complete conversion to bound IDO1 at micromolar concentrations of 3MI also demonstrated this binding occurs with a sub-micromolar Kd.


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Figure 5. Interaction of 3MI with IDO1. (A) Stopped flow UV-vis spectrometry of 3MI (30 µM) binding to IDO1 (1.4 µM) over 0.2 s. (B) Secondorder rate constant of hydrogen sulfide binding to IDO1 determined by stopped flow spectrometry. (C) Activity of IDO1 (500 nM) with L-tryptophan (200 µM) as substrate and activated by 3MI (500 nM) with and without glutathione (5 mM). (D) The activation parameters of 3MI and IDO1 (500 nM) with L-tryptophan (50 µM) as a substrate was fit to a Michaelis-Menten expression in blue. At higher concentrations of 3MI (the last four points), a single exponential curve (R2 = 0.9872) in purple was used to estimate an apparent Ki.

3MI was additionally shown to activate IDO1, inducing turnover even at nM concentrations (Figure 5C). Using 3MI, there was an initial burst of IDO1 activity, which leveled off after 500 s, well before substrate tryptophan was consumed (purple trace Figure 5C). Reduced glutathione was added to the activity assay solutions at nearly cellular concentrations (5 mM)57 to reduce any oxidized 3MIdisulfide products that may have formed, and this allowed for sustained 3MI-induced IDO1 activity (Figure 5C, blue trace). The Km of activation for 3MI, determined as with polysulfides, is 2.2 ± 0.7 µM (Figure 5D), and the kcat is 0.24 ± 0.04 µM. Above 5 µM 3MI, there was a steep inhibitory effect. The observed rate changes do not fit well to a simple substrate inhibition expression, and so a separate single exponential fit of the inhibitory region was used, showing an apparent Ki of 8 ± 3 µM. Despite its activating ability, 3MI did not cause the reduced ferrous form of IDO1 to accumulate (Figure S2C). Under low-oxygen conditions as described above, the 3MI-IDO1 complex formed and was quickly followed by the reappearance of the ferric enzyme. The 3MI-bound species then reaccumulated over 500 s though to a lesser extent than in the initial burst. This overall process occurred more

quickly when glutathione was added, producing a final 3MI-IDO1 complex that was stable for at least 1500 s (Figure S2C). The nature of 3MI-inhibition at high concentrations (>5 µM) was examined further with ferric-superoxo IDO1, the active tryptophan-oxidizing species. Ferric-superoxo IDO1 was generated using double-mixing stopped-flow spectrometry by mixing IDO1 pre-reduced with 2 mM sodium dithionite into aerobic buffer for 0.5 s. This ferric superoxo species was then mixed in the second push with 3MI that had been maintained in the reduced state using 5 mM glutathione (Figure S7). 3MI readily reacted with ferric-superoxo IDO1 to form ferric IDO1 without any observable intermediates. This was shown by a fast shift from the ferric-superoxo complex (λmax 415 nm) to the ferric (λmax 404 nm) that was completed nearly within the mixing time of the instrument (< 10 ms) even at the lowest concentration of 3MI useful for pseudo-first order reaction conditions (where 3MI must be at a large excess over IDO1). Following this fast conversion to ferric IDO1, there was a slower shift to the ferric 3MI-bound complex as indicated by the Soret shifting to 424 nm (as in Figure 5A). To account for both reactions, these traces were fit using a double exponential expression. The initial reaction of 3MI



with ferric-superoxo IDO1 thus showed a fast second-order reaction with an estimated rate constant > 107 M-1 s-1 (Figure S7). Since hydrogen sulfide and polysulfides are known to react with other heme proteins, the selectivity of 3MI for IDO1 was examined by mixing 3MI with several other heme proteins and observing their reactions using stopped-flow UV-vis spectrometry. 3MI had no effect on catalase even at 250 µM; whereas, 3MI did appear to reduce myoglobin and hemoglobin producing their oxygen-bound forms. 3MI was also able to reduce ferric cytochrome c to its distinctive ferrous form58 (Figure S8). The second-order rate constants of these reactions were determined under pseudo-first order conditions giving 6.6 x 103, 1.6 x 104, and 2.7 x 105 M-1 s-1 for myoglobin, hemoglobin and cytochrome c, respectively. These rates showed a general trend increasing with the FeIII/FeII reduction potential (Figure S9). However, despite having the lowest reduction potential this group, IDO1 reacted by binding to 3MI with a second-order rate constant of 1.7 x 106 M-1 s-1, exceeding that for the reduction reaction with the high potential cytochrome c by an order of magnitude, indicating selectivity of 3MI for IDO1. Unlike myoglobin and hemoglobin, the oxy-form of IDO1 was not observed due to the relatively slow reduction of IDO1 by 3MI (Figure S2). EPR Spectroscopy. The IDO1 complexes with polysulfide, sulfide and 3MI were characterized with electron paramagnetic resonance (EPR) spectroscopy at 11K. The ferric enzyme showed a mixture of rhombic high spin (g = 5.92, 5.48, and 1.99) and low spin components (g = 2.81, 2.27, 1.67) (Figure 6, black).59 Upon addition of 0.5 mM sodium disulfide to IDO1 (225 µM), the original ferric IDO1 signal was nearly eliminated, suggesting complete reduction to the EPR silent ferrous IDO1 within 1 min (Figure 6 in orange). After 30 min of incubation, the spectrum still showed very little signal, with only minor low spin rhombic ferric components that may represent disulfide-bound IDO1 or other species formed from transferring the sample in air to the EPR tube before freezing (Figure 6 in orange).60 The addition of 5 mM hydrogen sulfide to ferric IDO1 caused a significant decrease in the high spin signal and formation of a mixture of new rhombic low-spin signals with prominent features at g = 2.66, 2.26, and 1.77. This suggests the sulfide-bound species is a lowspin ferric hydrosulfido complex (Figure 6 in red). The reducing ability of hydrogen sulfide was evident by repeating this with a 30 min incubation prior to freezing wherein the signal was greatly diminished consistent with the conversion of IDO1 to the EPR-silent low spin ferrous form (Figure 6, red). EPR confirmed 3MI binds to IDO1 to form a species similar to that of hydrosulfido-IDO1 (Figure 6, purple). The EPR spectrum of 3MI-IDO1 showed a rhombic low spin species with g = 2.33, 2.22, and 1.92. The IDO1 signal remained in the presence of 3MI even after 30 min showing, as with the UV-vis experiments, that the EPRsilent ferrous IDO1 does not accumulate upon reaction with 3MI. Instead, the high spin ferric IDO1 signal (g = 5.92, 5.48, and 1.99) was partially recovered after 30 min incubation (Figure 6, purple). With the addition of 5 mM glutathione, however, the low-spin 3MI species was maintained (Figure 6, blue), consistent with glutathione regenerating a constant pool of reduced 3MI available to bind to IDO1. X-ray Absorption Near Edge Spectroscopy. K-edge XANES was used to characterize the complex formed by mixing polysulfides (1.3 mM) with IDO1 (0.96 mM) and flash freezing with liquid nitrogen within 1 min. This procedure resulted in complete

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Figure 6. EPR of IDO1. IDO1 (225 µM throughout) ferric as purified (black), sodium disulfide (500 µM) (orange), sodium sulfide (10 mM) (red), 3MI (100 µM) with 1% (v/v) DMSO (purple), and 3MI (100 µM) with 1% (v/v) DMSO and 5 mM glutathione (blue). Spectra were acquired at 11 K, 4.00 G modulation amplitude, 100 KHz modulation frequency, 6000 G sweep centered at 3200 G with 60 s sweep time, 9.37527 GHz microwave frequency, 0.6325 mW microwave power.


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Figure 7. Normalized Fe K-edge XANES of IDO1. (A) Comparison of ferric (red) and ferrous (blue) with 1.3 mM Na2S2 (black). (B) Comparison of IDO1 (960 µM) reduced to ferrous by 23 mM sodium dithionite (blue), as purified ferric (red), with 4.8 mM 3MI (green), and with 24 mM Na2S (yellow).

conversion of IDO1 to the ferrous form, with the resultant spectrum almost exactly overlaying with authentic ferrous IDO1 generated using sodium dithionite (Figure 7A). Similarly, combining IDO1 (0.96 mM) and sodium sulfide (20

mM) at pH 7.0 showed a distinct complex (Figure 7B, yellow line) intermediate between that of ferric IDO1 (Figure 7B, red line) and ferrous IDO1 (Figure 7B, blue line). This new species mirrored that formed with IDO1 and 3MI (4.8 mM with 8.6 mM glutathione) (Figure 7B, green line). These similar XANES features are consistent with EPR results showing both species are low spin complexes, further supporting that both are poised to donate an electron from their thiolate ligands to the ferric heme iron to produce the active ferrous enzyme. Discussion The results show that hydrogen disulfide and higher polysulfides bind rapidly to ferric human IDO1 leading to the reduction of the heme cofactor and its activation toward oxygen-binding and tryptophan dioxygenation (Figure 8). This scenario is evident from the dramatic shifts in the UV-vis spectrum of ferric IDO1 after mixing with polysulfides (Figure 2). Under nitrogen atmosphere, the initial polysulfide adduct converts mainly to reduced ferrous IDO1 (Figure S2). Further evidence from the EPR data showed that hydrogen disulfide induces a sharp decrease in signal, indicating formation of diamagnetic ferrous IDO1 (Figure 6). Similarly, the x-ray emission XANES data showed that disulfide treatment of ferric IDO1 produced a spectrum identical to authentic ferrous IDO1 (Figure 7). Polysulfide reduction is highly activating, inducing IDO1 turnover with L-tryptophan at previously reported maximal rates (~1-2 s-1) without any inhibitory effects (Figure 2). Polysulfide activation of IDO1 could be significant in a cellular context as the Km of polysulfide-IDO1-activation is determined here to be 2-10 µM (Figure 2 and Figure S1), within the range reported to exist in cells of from the nM61 to 2 µM62,44 and as high as 25 µM.43 These values are in line with the cellular concentrations of the more general class of sulfane sulfur species, such as protein-bound persulfides and glutathione persulfide (~ 100 µM), which readily interconvert with one another and have also shown potential as reducing agents of heme proteins.63-68 The contribution of polysulfides to reactivation of the cellular pool of ferric IDO1 has to be considered in the context of other potential reductive activation pathways.30, 32 The in vitro catalytic efficiencies of cytochrome b5 and cytochrome P450 reductase in reducing IDO1 exceed that of polysulfides (~ 108 M-1 s-1 compared to 1.7 ± 0.1 x 105 M-1 s-1 for polysulfides as in Figure 8), suggesting that these proteins play significant roles.50 However, the presence of ferric IDO1 in cells30 demonstrates that the autoxidative inactivation of

Figure 8. Reduction of inactive, ferric IDO1 (red) by polysulfides (sodium disulfide) or sodium sulfide and relevant kinetic parameters.



IDO1 effectively competes with these reduction pathways, raising the importance of determining the flux through each. Indeed, these multiple activating pathways may provide cellular versatility in regulating the kynurenine pathway. As with polysulfides, hydrogen sulfide was shown to bind to the ferric form of IDO1 causing reductive activation (Figure 3). Unlike polysulfides, however, hydrogen sulfide exhibited significantly weaker binding (Kd = 0.34 ± 0.02 mM) (Figure 3). The Km of sulfide-induced IDO1-activation is thus raised much higher relative to polysulfides (0.5 ± 0.1mM), far above the reported nM cellular concentration of hydrogen sulfide.55 Though hydrogen sulfide is not likely to alter IDO1 activity directly in vivo, recent evidence demonstrates many of the physiological effects of hydrogen sulfide may actually be the result of polysulfides that possess higher reactivity and cellular concentrations.52, 63 This linkage is made stronger by the heme protein-catalyzed production of polysulfides from hydrogen sulfide.35-36 IDO1 has here been shown to catalyze polysulfide biosynthesis using the polysulfide-selective fluorescent probe SSP4, linking hydrogen sulfide and polysulfide signaling with the kynurenine pathway through IDO1 (Figure S5). The reductive activation of IDO1 by polysulfides may fulfill a second purpose in preserving the heme-bound state of IDO1. Our previous work showed that IDO1 is dynamically bound to its heme cofactor and that heme-retention is significantly increased when IDO1 is in the ferrous state.26 Given the fast binding kinetics reported here, polysulfides are likely able to increase the cellular reservoir of heme-bound IDO1 through reduction to this more stable ferrous form. Due to the immunoregulatory role of IDO1, the pathways for polysulfide biosynthesis are of particular interest if they are upregulated in the immune response,69-71 including polysulfides produced from the reaction of hydrogen sulfide with immune response-derived nitric oxide and hypochlorite.72-77 The presence of polysulfides in the inflammatory environment is consistent with their reported

Figure 9. Proposed mechanism of catalytic reduction of IDO1 by 3MI. 3MI activates ferric IDO1 in a one-electron reduction. Two molecules of glutathione thiolate (GS) and molecular oxygen regenerate the reduced anion of 3MI and a further IDO1-activating superoxide molecule.

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antibiotic effects and the strong upregulation of persulfide-oxidizing enzymes in pathogenic bacteria.76, 78 Exogenous polysulfides delivered with N-acetyl cysteine persulfides can even reduce inflammation on an organismal scale.79 Polysulfides thus appear to be perfectly placed in the inflammatory environment to recruit and maintain active IDO1 to prevent excessive damage from immune responses. Polysulfides, along with hydrogen sulfide, are cytoprotective under oxidative stress,70, 80 which has spurred the invention of small molecules to deliver polysulfides in prodrug form,81-83 and these can now be considered for their potential to suppress the immune response through the polysulfide-activation of IDO1. These results also show that polysulfide biosynthesis could be a novel target for inhibition to stimulate the immune response against cancer and infections. The high reactivity of polysulfides likely limits their utility as a selective activator of the IDO1, so we sought a small molecule to replicate their activating effects with specificity for IDO1. 3-Mercaptoindole was chosen because it combines the features of tryptophan and hydrogen sulfide that together were seen to significantly enhance their binding to ferric IDO1, going from a hydrogen sulfide Kd ~ 0.4 mM without tryptophan to a Kd ~ 12 µM when L-tryptophan (800 µM) was present (Figure 4). Indeed, 3-mercaptoindole was observed to bind rapidly to ferric IDO1 (1.7 x 106 M-1 s-1) activating it for turnover even at nM concentrations (Figure 5). The reductive activation of IDO1 by 3MI is remarkably selective, as demonstrated by its much slower reaction with other heme proteins, for instance reducing cytochrome c more slowly than it binds to IDO1 by an order of magnitude (Figure S9). 3MI also activated IDO1 catalytically in the presence of glutathione (GSH). At low nM concentrations, 3MI was quickly depleted upon reaction with IDO1, but in the presence of cellularly relevant concentrations of GSH (~ 5 mM),57 the activating ability of 3MI was maintained over nearly an hour (Figure 5C). This observation supports a mechanism wherein the oxidized product of 3MI-activation of IDO1 is a 3MI-thiyl radical that can be recycled to the thiol by reacting with glutathione thiolate (present in the assay solution at 80 µM from 5 mM glutathione with a pKa 8.8 at pH 7.0).42 By comparison, sulfhydryl radical reacts at near diffusion-limited rates with thiolates (4.0 ± 0.6 x 109 M-1 s-1) at pH 7.0 to form a radical disulfide species that can then reduce oxygen to superoxide (4.0 ± 0.6 x 108 M-1 s-1) to afford the disulfide.84-85 In this way, the 3MI-thiyl radical can form an intermediate 3MI-GSH disulfide radical anion that would be expected to further react with oxygen to produce a mixed glutathione-3MI disulfide and superoxide. Superoxide itself is IDO1-activating,30 and the product 3MI-GSH disulfide can be reduced with another glutathione to free 3MI for additional rounds of IDO1-activation (Figure 9). The maximal turnover rate with 3MI activating IDO1 (~ 0.16 s1 ) is lower than that achieved with polysulfides and ascorbate-methylene blue (1-2 s-1).49 Since the activity of IDO1 in cells is known to be sensitive to the concentration of cytochrome b5, it is likely that a significant portion of IDO1 exists in the inactive ferric form in vivo.30 Thus, the ability to maintain a constant IDO1 activity in cells could have pronounced effects perhaps even at turnover rates shown with 3MI. This limitation in the activating ability of 3MI is likely to be caused by its side-reactivity with the intermediate, ferric-superoxo form of IDO1 (Figure S7), and efforts to modify 3MI to prevent this are currently underway. An attractive feature, however, is that the sustained activating ability of 3MI in the presence of glutathione shows that these secondary reactions are easily reversible, something


Page 9 of 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


that would not be the case if 3MI were oxygenated on its indole core. 3MI may thus serve as the basis for a novel class of IDO1-activating agents. Remarkably, by reacting with both glutathione and IDO1, 3MI allows IDO1 to access the vast reducing power of the glutathione system in cells, holding it in the active state low nM concentrations. (Figure 9). This has a further benefit in that 3MI may be delivered to cells in the oxidized and less reactive disulfide form from which it can then be reduced to the active thiol in the reducing environment of the cytoplasm.86 In conclusion, this work shows that polysulfides bind avidly to ferric IDO1 and activate it by reduction to the ferrous state. Since autoxidation is one of the key regulatory mechanisms impeding the activity of IDO1, the ability to reduce the heme-iron in IDO1 at physiologically relevant concentrations implicates polysulfides in the replenishment and sustained activity of IDO1. Additionally, we have discovered that a small molecule, 3-mercaptoindole, replicates the activating effects of polysulfides with selectivity for IDO1. This represents the first known selective IDO1 activator for tryptophan oxidation and provides a potentially powerful new tool in fighting autoimmune disorders and many other diseases affected by hyperactive immune responses. Experimental Section Materials. All materials were provided by Sigma-Aldrich unless otherwise indicated. Expression and Purification of IDO1. The gene for full-length human IDO1 was inserted into pET28a(+) between the NdeI and XhoI cut sites to add an N-terminal histidine tag (Genscript). This was expressed in E. coli BL21(DE3) (New England Biolabs). These were grown at 37 °C in baffled 1 L Fernbach flasks using 1 L of lysogeny broth (Research Products International) supplemented with 34 µg/mL kanamycin (Research Products International). Once the optical density at 600 nm reached 0.5, cultures were brought to room temperature (~ 22 °C) and were supplemented with 50 µM ferric chloride, 1 mM isopropyl β-D-1-thiogalactopyranoside, and 0.5 mM δ-aminolevulinic acid (Frontier Scientific). After 12 h of room temperature expression, the cultures were brought to 4 °C for a further 12 h without stirring. The cells were collected with centrifugation (5000 xg) for 30 min at 4 °C and stored at -20 °C. The cells were disrupted at 4 °C using sonication (Branson) in 50 mM sodium phosphate pH 7.4 with 20 mM imidazole and 200 mM sodium chloride. Insoluble debris was removed with centrifugation (5000 xg) for 60 min at 4 °C. The supernatant was passed over a 5 mL FF HisTrap column (GE Healthcare). The bound protein was washed with five column volumes of the same buffer and was then eluted by increasing the concentration of imidazole to 500 mM. The collected protein was concentrated at 4 °C using polyethersulfone centrifugal filters with 10 kDa cutoff (GE Healthcare) and was then exchanged into 100 mM sodium phosphate pH 7.0 with 200 mM sodium chloride using a DG-10 size exclusion column (Bio-Rad). The protein was then flash frozen with liquid nitrogen and stored at -80 °C. Activity Assays. UV-vis spectra were recorded using a Hewlett-Packed 8453 diode array spectrophotometer. Activity assays were done at room temperature using 100 mM sodium phosphate pH 7.0 with 500 nM IDO1 (ε404 nm = 172 mM-1 cm-1)48 and 50 µM L-tryptophan. These were initiated by addition of IDO1, and activity was monitored at 321 nm where product N-formylkynurenine absorbs (ε321 nm = 3750 M-1 cm-1).48 Sodium sulfide was prepared daily in a 1M stock solution using 0.1 M sodium hydroxide, and activation of IDO1 was tested at various concentrations ranging from 100 µM to 20 mM. Hydrogen disulfide, hydrogen trisulfide, and hydrogen tetrasulfide (Dojindo Molecular Technologies) were prepared in 10 mM stock solutions using 0.1 M sodium hydroxide in which they are weakly stable and maintain their average chain length.64 These were stored at – 80 °C and used only once after thawing. They were each tested from 40 nM to 25.6 µM. 3-Mercaptoindole was prepared daily in a 100 mM stock solution in dimethyl sulfoxide (DMSO), and this was serially diluted

to maintain a consistent final concentration of 1% (v/v) DMSO in each assay. To maintain activity, 5 mM reduced glutathione was added to some of these from a 0.5 M stock solution. 3MI was tested from 100 nM to 10 µM. The ability of each compound to serve as a reducing substrate for IDO1 was fit using a Michaelis-Menten equation, and in the cases of hydrogen sulfide, an additional term of inhibition was added: 𝑘𝑐𝑎𝑡 × [𝑎𝑐𝑡𝑖𝑣𝑎𝑡𝑜𝑟] 𝑣= 1 + [𝑎𝑐𝑡𝑖𝑣𝑎𝑡𝑜𝑟] 𝐾𝑚 + 12 4 [𝑎𝑐𝑡𝑖𝑣𝑎𝑡𝑜𝑟]5 𝐾𝑖 The activity and inhibition caused by 3MI did not fit well to a simple substrate inhibition expression and was instead fit at higher concentrations of 3MI using a single exponential equation to describe an apparent Ki at halfactivity derived similarly to a half-life. Reduction assays were done using IDO1 (5 µM) in 100 mM sodium phosphate pH 7.0 placed in a 1 mL quartz cuvette with a septum (Starna) and flushed with nitrogen through a needle to remove molecular oxygen from the headspace. Sodium sulfide at 100 mM in 100 mM sodium hydroxide was added with a syringe to a final concentration of 10 mM. Spectra were recorded every 30 s over 30 min at room temperature. Binding Assay. Stopped flow experiments were done using a Hi-Tech SF-61DX2 double mixing instrument with a 10 mm path length cuvette equipped with a Fisher Scientific Isotemp 1016S water bath to maintain temperature at 25 ± 1 °C. The average of at least five shots were used for each experiment. IDO1 (2 µM, final 1 µM) was prepared in one syringe in 100 mM sodium phosphate pH 7.0. This was mixed with analytes prepared in the other syringe: sodium sulfide concentration was varied by addition of 100 mM stock into 10 mM sodium hydroxide; polysulfide concentrations were varied using 1 mM stocks added to 10 mM sodium hydroxide; and 3MI was added from stock solutions in DMSO (maintaining a final 5% (v/v) DMSO) added to 100 mM sodium phosphate pH 7.0 with 5 mM glutathione (final glutathione 2.5 mM). Pseudo first-order kinetics were obtained fitting the decay in the absorbance of IDO1 at 404 nm to a single exponential in the cases of binding of hydrogen sulfide and 3MI; for polysulfides that had significant absorption at 404 nm, the increase in the absorption of polysulfide-bound IDO1 at 425 nm was fit to a single exponential plus a linear fit, the latter of which accounts for polysulfide scattering that occurs upon mixing into neutral buffer even in the absence of IDO1. It must be noted that polysulfides interconvert in aqueous solution resulting in a distribution of polysulfides depending on pH.44, 51 The species reported here are those of starting polysulfide chain length as provided by the supplier (Dojindo). The pKa1 for dihydrogen disulfide, trisulfide, and tetrasulfide (5.0, 4.2 and 3.8, respectively) are low enough that at the tested pH of 7.0 the coordinating ligand is likely to be the mono-deprotonated form (HSn–), and it is possible given the pKa2 (9.7, 7.5, and 6.3) that the doubly deprotonated polysulfides are responsible for binding as well.87 The concentrations reported here are those of total polysulfide from stock sodium polysulfide solutions regardless of protonation. The effect of L-tryptophan on sulfide binding was tested by including Ltryptophan (final concentrations of 50 to 800 µM) in solution with IDO1 in 100 mM sodium phosphate pH 7.0 and mixing into sodium sulfide in 10 mM sodium hydroxide. The resultant spectra were fit to a series of linear combinations of as-purified IDO1 and sulfide-bound IDO1 using Matlab, which were then fit to a binding curve to obtain the Kd of sulfide and IDO1 at various concentrations of L-tryptophan. Bovine catalase and hemoglobin, and horse heart cytochrome c and myoglobin were all obtained from Sigma Aldrich, resuspended in 100 mM sodium phosphate pH 7.0, and used without further purification. These were mixed with various concentrations of 3MI made from stock solutions in DMSO in the same buffer with 5 mM glutathione (final 2.5 mM) and final 5% (v/v) DMSO. Pseudo first-order kinetics were obtained by fitting the change in absorbance of myoglobin at 409 nm, hemoglobin at 404 nm, and cytochrome c at 550 nm to single exponentials. Reduction Assays. IDO1 (8 µM) was placed in 100 mM sodium phosphate pH 7.0 in sealed cuvettes using buffer that had been vacuum-degassed



just prior to use. Additionally, the vessel headspace was flushed with nitrogen. Using a syringe, sodium sulfide (10 mM), sodium hydrodisulfide (10 µM), 3MI (100 µM with final 1% DMSO v/v), and 3MI (100 µM with final 1% DMSO v/v) with glutathione (5 mM) were added through a septum. Reduction was monitored using UV-vis spectrometry. Rates of reduction were estimated using kinetic studio software (HiTech) fitting the increases in absorbance at 560 nm to a single exponential expression. For hydrogen sulfide this absorbance was baseline-corrected by subtracting the corresponding absorbance at 800 nm; for hydrogen disulfide, this was instead corrected using absorbance at 600 nm because of the large amount of scattering. Reaction with Ferric Superoxo IDO1. The reaction of 3MI with ferricsuperoxo IDO1, produced as previously described,29 was tested using double mixing stopped flow. Briefly, IDO1 (1.5 µM) was added to 2 mM sodium dithionite in one syringe, and in the first push this was mixed into aerobic buffer and allowed to incubate for 0.5 s after which a second push mixed this into sodium sulfide (10 mM) or 3MI (20 µM) with final 5%(v/v) DMSO and 2.5 mM glutathione. All buffers used were 100 mM sodium phosphate pH 7.0. The reaction was followed by fitting the change in absorbance of IDO1 at 404 nm to a double exponential expression following the initial burst of ferric-superoxo (λmax 415 nm) converting to ferric IDO1 (λmax 404 nm) and a final slower binding of ferric IDO1 to 3MI (λmax 424 nm). Polysulfide Production Assay. An Agilent Technologies Cary Eclipse Fluorescent Spectrophotometer was used to test fluorescence. Polysulfide production from IDO1 and hydrogen sulfide was tested by incubating IDO1 (10 µM), sodium sulfide (2 mM), and a sulfane sulfur fluorescent probe SSP4 (100 µM) (Dojindo Molecular Technologies) in the dark at room temperature for 1 hr. From this 10 µL were added to 90 µL of deionized water and tested for fluorescence with excitation at 482 nm with emission scanning from 500 to 600 nm. Electron Paramagnetic Resonance. Samples for EPR experiments were prepared using IDO1 with a final concentration of 225 µM in each. The buffer for these was 60 mM sodium phosphate pH 7.0 with 40% (v/v) glycerol. Samples with 3MI were made using a 100 mM stock in DMSO (final 1% v/v), and one of these additionally had final 5 mM glutathione. Samples with sodium sulfide (5 mM) were prepared using a 0.5 M stock in 100 mM sodium hydroxide. Those with polysulfide used hydrogen disulfide (500 µM) (Dojindo Molecular Technologies) and were prepared from a 10 mM polysulfide stock in 10 mM sodium hydroxide. For each set of conditions, two samples were made with one being frozen within 1 min of mixing and the second after 30 min of mixing. EPR experiments were recorded on a Bruker Elexsys 580 X-band CWEPR system. The spectra were obtained at 14 K using liquid helium with an Oxford Instruments EPR cryostat with an Oxford ITC 503S temperature controller, and the following parameters were used: 4.00 G modulation amplitude, 100 KHz modulation frequency, 6000 G sweep centered at 3200 G with 60 s sweep time, 9.37527 GHz microwave frequency, 0.6325 mW microwave power. XANES. Samples for XANES were prepared using IDO1 exchanged into 60 mM sodium phosphate pH 7.0 with 40% (v/v) glycerol and concentrated to 960 µM. Samples (~ 40 uL) were frozen in liquid nitrogen within 1 min of mixing. Ferric IDO1 was used without any further additions. Ferrous IDO1 was made by addition of 0.5 M sodium dithionite to 23 mM. IDO1 with hydrogen disulfide was made by addition of 10 mM hydrogen disulfide in 10 mM sodium hydroxide to 1.3 mM. IDO1 with sodium sulfide was prepared by addition of 0.5 M sodium sulfide in 100 mM sodium hydroxide to 24 mM. IDO1 with 3MI was prepared by addition of 0.1 M 3MI in DMSO to 4.8 mM with final 4.8 % DMSO (v/v) along with 0.5 M glutathione to 8.6 mM. Fe K-edge x-ray absorption spectra were collected at the Advanced Photon Source of Argonne National Lab on the bending magnet beamline 20BM. Data were recorded as fluorescence excitation spectra using a 13-element (Ge) energy-resolving detector, and internal calibration of the incident energy was accomplished via the concomitant collection of a transmission spectrum of iron foil with the inflection point assigned to 7112 eV. To minimize x-ray-induced damage, the beam was both defocused (1 x 6 mm2) and detuned by 15%, while samples were maintained at ~ 20 K during data collection using a helium gas-exchange cryostat. Scans were additionally monitored for reductive shifts, and no more than 5 scans were accumulated on

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any one spot. Athena software was used for pre- and post-edge corrections, background removal, averaging, and normalization.88

ASSOCIATED CONTENT Supporting Information Additional figures as described in the text can be found in the supporting information. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author * [email protected] ORCID Micah T. Nelp: 0000-0002-9716-5842 Vincent Zheng: 0000-0002-8232-3190 Katherine M. Davis: 0000-0002-0258-8907 Katherine J. E. Stiefel: 0000-0002-9114-7918 John T. Groves: 0000-0002-9944-5899

Author Contributions The data was gathered and the manuscript was written through contributions of all authors.

Funding Sources Support for this work was provided by the National Institutes of Health (2R37 GM036298). K.M.D. is supported by an NIH K99 Pathway to Independence Award (1K99GM129460-01). M.T.N. was supported in part by the Eric and Wendy Schmidt Transformative Technology Fund. K.J.E.S. was supported in part by the Lewis-Siegler Institute for Integrative Genomics.

References 1. Yamamoto, S.; Hayaishi, O., Tryptophan pyrrolase of rabbit intestine D-and L-tryptophan-cleaving enzyme or enzymes. J. Biol. Chem. 1967, 242 (22), 5260-5266. 2. Shimizu, T.; Nomiyama, S.; Hirata, F.; Hayaishi, O., Indoleamine 2, 3-dioxygenase. Purification and some properties. J. Biol. Chem. 1978, 253 (13), 4700-4706. 3. Ball, H. J.; Yuasa, H. J.; Austin, C. J.; Weiser, S.; Hunt, N. H., Indoleamine 2, 3-dioxygenase-2; a new enzyme in the kynurenine pathway. Int. J. Biochem. Cell Biol. 2009, 41 (3), 467-471. 4. Badawy, A. A.-B., Kynurenine pathway of tryptophan metabolism: regulatory and functional aspects. Int. J. Tryptophan Res. 2017, 10, 1-20. 5. Taylor, M. W.; Feng, G., Relationship between interferongamma, indoleamine 2, 3-dioxygenase, and tryptophan catabolism. The FASEB J. 1991, 5 (11), 2516-2522. 6. Mellor, A. L.; Munn, D. H., IDO expression by dendritic cells: tolerance and tryptophan catabolism. Nat. Rev. Immunol. 2004, 4 (10), 762774. 7. Lewis-Ballester, A.; Batabyal, D.; Egawa, T.; Lu, C.; Lin, Y.; Marti, M. A.; Capece, L.; Estrin, D. A.; Yeh, S.-R., Evidence for a ferryl intermediate in a heme-based dioxygenase. Proc. Natl. Acad. Sci. 2009, 106 (41), 17371-17376. 8. Basran, J.; Efimov, I.; Chauhan, N.; Thackray, S. J.; Krupa, J. L.; Eaton, G.; Griffith, G. A.; Mowat, C. G.; Handa, S.; Raven, E. L., The mechanism of formation of N-formylkynurenine by heme dioxygenases. J. Am. Chem. Soc. 2011, 133 (40), 16251-16257. 9. Capece, L.; Lewis-Ballester, A.; Yeh, S.-R.; Estrin, D. A.; Marti, M. A., Complete reaction mechanism of indoleamine 2, 3-dioxygenase as


Page 11 of 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


revealed by QM/MM simulations. J. Phys. Chem. B 2012, 116 (4), 14011413. 10. Shin, I.; Ambler, B. R.; Wherritt, D.; Griffith, W. P.; Maldonado, A. C.; Altman, R. A.; Liu, A., Stepwise O-Atom Transfer in Heme-Based Tryptophan Dioxygenase: Role of Substrate Ammonium in Epoxide Ring Opening. J. Am. Chem. Soc. 2018, 140 (12), 4372-4379. 11. Dai, X.; Zhu, B. T., Indoleamine 2, 3-dioxygenase tissue distribution and cellular localization in mice: implications for its biological functions. J. Histochem. Cytochem. 2010, 58 (1), 17-28. 12. Munn, D. H.; Mellor, A. L., Indoleamine 2, 3 dioxygenase and metabolic control of immune responses. Trends Immunol. 2013, 34 (3), 137143. 13. Munn, D. H.; Zhou, M.; Attwood, J. T.; Bondarev, I.; Conway, S. J.; Marshall, B.; Brown, C.; Mellor, A. L., Prevention of allogeneic fetal rejection by tryptophan catabolism. Science 1998, 281 (5380), 1191-1193. 14. Uyttenhove, C.; Pilotte, L.; Théate, I.; Stroobant, V.; Colau, D.; Parmentier, N.; Boon, T.; Van den Eynde, B. J., Evidence for a tumoral immune resistance mechanism based on tryptophan degradation by indoleamine 2, 3-dioxygenase. Nat. Med. 2003, 9 (10), 1269-1274. 15. Gautam, U. S.; Foreman, T. W.; Bucsan, A. N.; Veatch, A. V.; Alvarez, X.; Adekambi, T.; Golden, N. A.; Gentry, K. M.; Doyle-Meyers, L. A.; Russell-Lodrigue, K. E., In vivo inhibition of tryptophan catabolism reorganizes the tuberculoma and augments immune-mediated control of Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. 2018, 115 (1), E62-E71. 16. Hornyák, L.; Dobos, N.; Koncz, G.; Karányi, Z.; Páll, D.; Szabó, Z.; Halmos, G.; Székvölgyi, L., The Role of indoleamine-2, 3-Dioxygenase in Cancer Development, Diagnostics, and Therapy. Front. Immunol. 2018, 9 (151). 17. Orabona, C.; Mondanelli, G.; Pallotta, M. T.; Carvalho, A.; Albini, E.; Fallarino, F.; Vacca, C.; Volpi, C.; Belladonna, M. L.; Berioli, M. G., Deficiency of immunoregulatory indoleamine 2, 3-dioxygenase 1 in juvenile diabetes. JCI insight 2018, 3 (6), 96244. 18. Mondanelli, G.; Iacono, A.; Carvalho, A.; Orabona, C.; Volpi, C.; Pallotta, M. T.; Matino, D.; Esposito, S.; Grohmann, U., Amino acid metabolism as drug target in autoimmune diseases. Autoimmun. Rev. 2019, 334-348. 19. Ketelhuth, D. F., The immunometabolic role of indoleamine 2 3dioxygenase in atherosclerotic cardiovascular disease: immune homeostatic mechanisms in the artery wall. Cardiovasc. Res. 2019, cvz067. 20. Szántó, S.; Koreny, T.; Mikecz, K.; Glant, T. T.; Szekanecz, Z.; Varga, J., Inhibition of indoleamine 2, 3-dioxygenase-mediated tryptophan catabolism accelerates collagen-induced arthritis in mice. Arthrit. Res. Ther. 2007, 9 (3), R50. 21. Albini, E.; Rosini, V.; Gargaro, M.; Mondanelli, G.; Belladonna, M. L.; Pallotta, M. T.; Volpi, C.; Fallarino, F.; Macchiarulo, A.; Antognelli, C., Distinct roles of immunoreceptor tyrosine-based motifs in immunosuppressive indoleamine 2, 3-dioxygenase 1. J. Cell. Mol. Med. 2017, 21 (1), 165-176. 22. Orabona, C.; Pallotta, M. T.; Volpi, C.; Fallarino, F.; Vacca, C.; Bianchi, R.; Belladonna, M. L.; Fioretti, M. C.; Grohmann, U.; Puccetti, P., SOCS3 drives proteasomal degradation of indoleamine 2, 3-dioxygenase (IDO) and antagonizes IDO-dependent tolerogenesis. Proc. Natl. Acad. Sci. 2008, 105 (52), 20828-20833. 23. Fujigaki, H.; Saito, K.; Lin, F.; Fujigaki, S.; Takahashi, K.; Martin, B. M.; Chen, C. Y.; Masuda, J.; Kowalak, J.; Takikawa, O., Nitration and inactivation of IDO by peroxynitrite. J. Immunol. 2006, 176 (1), 372-379. 24. Thomas, S. R.; Terentis, A. C.; Cai, H.; Takikawa, O.; Levina, A.; Lay, P. A.; Freewan, M.; Stocker, R., Post-translational regulation of human indoleamine 2, 3-dioxygenase activity by nitric oxide. J. Biol. Chem. 2007, 282 (33), 23778-23787. 25. Lewis-Ballester, A.; Karkashon, S.; Batabyal, D.; Poulos, T. L.; Yeh, S.-R., Inhibition Mechanisms of Human Indoleamine 2, 3 Dioxygenase 1. J. Am. Chem. Soc. 2018, 140, 8518-8525. 26. Nelp, M. T.; Kates, P. A.; Hunt, J. T.; Newitt, J. A.; Balog, A.; Maley, D.; Zhu, X.; Abell, L.; Allentoff, A.; Borzilleri, R.; Lewis, H. A.; Lin, Z.; Seitz, S. P.; Yan, C.; Groves, J. T., Immune-modulating enzyme indoleamine 2, 3-dioxygenase is effectively inhibited by targeting its apoform. Proc. Natl. Acad. Sci. 2018, 115 (13), 3249-3254.

27. Ortiz-Meoz, R. F.; Wang, L.; Matico, R.; Rutkowska, A.; De la Rosa, M.; Bedard, S.; Midgett, R.; Strohmer, K.; Thomson, D.; Zhang, C., Characterization of novel inhibition of indoleamine 2, 3-dioxygenase by targeting its apo form. bioRxiv 2018, 324947. 28. Chen, A. Y.; Adamek, R. N.; Dick, B. L.; Credille, C. V.; Morrison, C. N.; Cohen, S. M., Targeting Metalloenzymes for Therapeutic Intervention. Chem. Rev. 2018, 119 (2), 1323-1455. 29. Booth, E. S.; Basran, J.; Lee, M.; Handa, S.; Raven, E. L., Substrate oxidation by indoleamine 2, 3-dioxygenase: Evidence for a common reaction mechanism. J. Biol. Chem. 2015, 290 (52), 30924-30930. 30. Maghzal, G. J.; Thomas, S. R.; Hunt, N. H.; Stocker, R., Cytochrome b5, not superoxide anion radical, is a major reductant of indoleamine 2, 3-dioxygenase in human cells. J. Biol. Chem. 2008, 283 (18), 12014-12025. 31. Thomas, S. R.; Salahifar, H.; Mashima, R.; Hunt, N. H.; Richardson, D. R.; Stocker, R., Antioxidants inhibit indoleamine 2, 3dioxygenase in IFN-γ-activated human macrophages: posttranslational regulation by pyrrolidine dithiocarbamate. J. Immunol. 2001, 166 (10), 6332-6340. 32. Vottero, E.; Mitchell, D. A.; Page, M. J.; MacGillivray, R. T.; Sadowski, I. J.; Roberge, M.; Mauk, A. G., Cytochrome b 5 is a major reductant in vivo of human indoleamine 2, 3-dioxygenase expressed in yeast. FEBS Lett. 2006, 580 (9), 2265-2268. 33. Kolawole, A. O.; Hixon, B. P.; Dameron, L. S.; Chrisman, I. M.; Smirnov, V. V., Catalytic activity of human indoleamine 2, 3-dioxygenase (hIDO1) at low oxygen. Arch. Biochem. Biophys. 2015, 570, 47-57. 34. Vitvitsky, V.; Yadav, P. K.; Kurthen, A.; Banerjee, R., Sulfide oxidation by a noncanonical pathway in red blood cells generates thiosulfate and polysulfides. J. Biol. Chem. 2015, 290 (13), 8310-8320. 35. Bostelaar, T.; Vitvitsky, V.; Kumutima, J.; Lewis, B. E.; Yadav, P. K.; Brunold, T. C.; Filipovic, M.; Lehnert, N.; Stemmler, T. L.; Banerjee, R., Hydrogen sulfide oxidation by myoglobin. J. Am. Chem. Soc. 2016, 138 (27), 8476-8488. 36. Vitvitsky, V.; Yadav, P. K.; An, S.; Seravalli, J.; Cho, U.-S.; Banerjee, R., Structural and mechanistic insights into hemoglobin-catalyzed hydrogen sulfide oxidation and the fate of polysulfide products. J. Biol. Chem. 2017, 292 (13), 5584-5592. 37. Mittra, K.; Singha, A.; Dey, A., Mechanism of reduction of ferric porphyrins by sulfide: identification of a low spin FeIII–SH intermediate. Inorg. Chem. 2017, 56 (7), 3916-3925. 38. Boubeta, F. M.; Bieza, S. A.; Bringas, M.; Estrin, D. A.; Boechi, L.; Bari, S. E., Mechanism of Sulfide Binding by Ferric Hemeproteins. Inorg. Chem. 2018, 57, 7591-7600. 39. Jensen, B.; Fago, A., Reactions of ferric hemoglobin and myoglobin with hydrogen sulfide under physiological conditions. J. Inorg. Biochem. 2018, 182, 133-140. 40. Matsui, T.; Sugiyama, R.; Sakanashi, K.; Tamura, Y.; Iida, M.; Nambu, Y.; Higuchi, T.; Suematsu, M.; Ikeda-Saito, M., Hydrogen sulfide bypasses the rate-limiting oxygen activation of heme oxygenase. J. Biol. Chem. 2018, 293 (43), 16931-16939. 41. Filipovic, M. R.; Zivanovic, J.; Alvarez, B.; Banerjee, R., Chemical biology of H2S signaling through persulfidation. Chem. Rev. 2017, 118 (3), 1253-1337. 42. Mishanina, T. V.; Libiad, M.; Banerjee, R., Biogenesis of reactive sulfur species for signaling by hydrogen sulfide oxidation pathways. Nat. Chem. Biol. 2015, 11 (7), 457-464. 43. Kimura, Y.; Mikami, Y.; Osumi, K.; Tsugane, M.; Oka, J.-i.; Kimura, H., Polysulfides are possible H2S-derived signaling molecules in rat brain. The FASEB J. 2013, 27 (6), 2451-2457. 44. Koike, S.; Kawamura, K.; Kimura, Y.; Shibuya, N.; Kimura, H.; Ogasawara, Y., Analysis of endogenous H2S and H2Sn in mouse brain by high-performance liquid chromatography with fluorescence and tandem mass spectrometric detection. Free Radical Biol. Med. 2017, 113, 355-362. 45. Vitvitsky, V.; Miljkovic, J. L.; Bostelaar, T.; Adhikari, B.; Yadav, P. K.; Steiger, A. K.; Torregrossa, R.; Pluth, M. D.; Whiteman, M.; Banerjee, R.; Filipovic, M. R., Cytochrome c Reduction by H2S Potentiates Sulfide Signaling. ACS Chem. Biol. 2018, 13 (8), 2300-2307.



46. Fukuto, J. M.; Ignarro, L. J.; Nagy, P.; Wink, D. A.; Kevil, C. G.; Feelisch, M.; Cortese-Krott, M. M.; Bianco, C. L.; Kumagai, Y.; Hobbs, A. J., Biological hydropersulfides and related polysulfides–a new concept and perspective in redox biology. FEBS Lett. 2018, 592 (12), 2140-2152. 47. Efimov, I.; Basran, J.; Sun, X.; Chauhan, N.; Chapman, S. K.; Mowat, C. G.; Raven, E. L., The mechanism of substrate inhibition in human indoleamine 2, 3-dioxygenase. J. Am. Chem. Soc. 2012, 134 (6), 3034-3041. 48. Papadopoulou, N. D.; Mewies, M.; McLean, K. J.; Seward, H. E.; Svistunenko, D. A.; Munro, A. W.; Raven, E. L., Redox and spectroscopic properties of human indoleamine 2, 3-dioxygenase and a His303Ala variant: implications for catalysis. Biochemistry 2005, 44 (43), 14318-14328. 49. Sono, M., The roles of superoxide anion and methylene blue in the reductive activation of indoleamine 2, 3-dioxygenase by ascorbic acid or by xanthine oxidase-hypoxanthine. J. Biol. Chem. 1989, 264 (3), 1616-1622. 50. Pearson, J. T.; Siu, S.; Meininger, D. P.; Wienkers, L. C.; Rock, D. A., In vitro modulation of cytochrome P450 reductase supported indoleamine 2, 3-dioxygenase activity by allosteric effectors cytochrome b 5 and methylene blue. Biochemistry 2010, 49 (12), 2647-2656. 51. Kamyshny, A.; Goifman, A.; Gun, J.; Rizkov, D.; Lev, O., Equilibrium distribution of polysulfide ions in aqueous solutions at 25 C: a new approach for the study of polysulfides' equilibria. Environ. Sci. Technol. 2004, 38 (24), 6633-6644. 52. Yuan, S.; Shen, X.; Kevil, C. G., Beyond a gasotransmitter: hydrogen sulfide and polysulfide in cardiovascular health and immune response. Antioxid. Red. Signal. 2017, 27 (10), 634-653. 53. Gutfreund, H., Kinetics for the life sciences: receptors, transmitters and catalysts. Cambridge University Press: Cambridge, 1995. 54. Chen, W.; Liu, C.; Peng, B.; Zhao, Y.; Pacheco, A.; Xian, M., New fluorescent probes for sulfane sulfurs and the application in bioimaging. Chem. Sci. 2013, 4 (7), 2892-2896. 55. Vitvitsky, V.; Kabil, O.; Banerjee, R., High turnover rates for hydrogen sulfide allow for rapid regulation of its tissue concentrations. Antioxid. Red. Signal. 2012, 17 (1), 22-31. 56. Heidemann, R.; Lütkemeyer, D.; Büntemeyer, H.; Lehmann, J., Effects of dissolved oxygen levels and the role of extra-and intracellular amino acid concentrations upon the metabolism of mammalian cell lines during batch and continuous cultures. Cytotechnology 1998, 26 (3), 185197. 57. Montero, D.; Tachibana, C.; Winther, J. R.; Appenzeller-Herzog, C., Intracellular glutathione pools are heterogeneously concentrated. Redox Biol. 2013, 1 (1), 508-513. 58. Macyk, J.; van Eldik, R., Kinetics of the reduction of cytochrome c by [Fe II (edta)(H 2 O)] 2−: outer-sphere vs. inner-sphere electron transfer mechanisms. Dalton Trans. 2003, (13), 2704-2709. 59. Sono, M.; Dawson, J. H., Extensive studies of the heme coordination structure of indoleamine 2, 3-dioxygenase and of tryptophan binding with magnetic and natural circular dichroism and electron paramagnetic resonance spectroscopy. Biochim. Biohys. Acta 1984, 789 (2), 170-187. 60. Davydov, R. M.; Chauhan, N.; Thackray, S. J.; Anderson, J. R.; Papadopoulou, N. D.; Mowat, C. G.; Chapman, S. K.; Raven, E. L.; Hoffman, B. M., Probing the ternary complexes of indoleamine and tryptophan 2, 3-dioxygenases by cryoreduction EPR and ENDOR spectroscopy. J. Am. Chem. Soc. 2010, 132 (15), 5494-5500. 61. Bibli, S.-I.; Luck, B.; Zukunft, S.; Wittig, J.; Chen, W.; Xian, M.; Papapetropoulos, A.; Hu, J.; Fleming, I., A selective and sensitive method for quantification of endogenous polysulfide production in biological samples. Redox Biol. 2018, 18, 295-304. 62. Kimura et al. (2013) reported polysulfide concentrations to be 25 nmol / g wet brain tissue. Assuming a density of 1 g / mL for brain tissue gives 25 µM polysulfide. A recent report from the same group (Koike, et al., 2018) used a more sensitive method in which concentrations were reported as µmol / g protein. The brain samples of 0.2 g supplied 16 mg protein (2 mL of 7-9 mg/ mL protein after homogenization). There was 0.08 g protein / g brain tissue (16 mg protein / 0.2 g brain tissue), giving a conversion factor to M concentration, assuming a density of 1 g / mL for brain tissue. Hydrogen disulfide at 0.026 ± 0.002 µmol / g protein is thus 2.08 ± 0. 16 nmol / mL brain, or about 2 µM.

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63. Toohey, J. I., Sulfur signaling: is the agent sulfide or sulfane? Anal. Biochem. 2011, 413 (1), 1-7. 64. Koike, S.; Ogasawara, Y., Sulfur atom in its bound state is a unique element involved in physiological functions in mammals. Molecules 2016, 21 (12), E1753. 65. Ida, T.; Sawa, T.; Ihara, H.; Tsuchiya, Y.; Watanabe, Y.; Kumagai, Y.; Suematsu, M.; Motohashi, H.; Fujii, S.; Matsunaga, T., Reactive cysteine persulfides and S-polythiolation regulate oxidative stress and redox signaling. Proc. Natl. Acad. Sci. 2014, 111 (21), 7606-7611. 66. Galardon, E.; Huguet, F.; Herrero, C.; Ricoux, R.; Artaud, I.; Padovani, D., Reactions of persulfides with the heme cofactor of oxidized myoglobin and microperoxidase 11: reduction or coordination. Dalton Trans. 2017, 46 (24), 7939-7946. 67. Bailey, T. S.; Zakharov, L. N.; Pluth, M. D., Understanding hydrogen sulfide storage: probing conditions for sulfide release from hydrodisulfides. J. Am. Chem. Soc. 2014, 136 (30), 10573-10576. 68. Yadav, P. K.; Martinov, M.; Vitvitsky, V.; Seravalli, J.; Wedmann, R.; Filipovic, M. R.; Banerjee, R., Biosynthesis and reactivity of cysteine persulfides in signaling. J. Am. Chem. Soc. 2015, 138 (1), 289-299. 69. Kimura, Y.; Toyofuku, Y.; Koike, S.; Shibuya, N.; Nagahara, N.; Lefer, D.; Ogasawara, Y.; Kimura, H., Identification of H 2 S 3 and H 2 S produced by 3-mercaptopyruvate sulfurtransferase in the brain. Sci. Rep. 2015, 5, 14774. 70. Kimura, H., Signaling molecules: hydrogen sulfide and polysulfide. Antioxid. Redox Signal. 2015, 22 (5), 362-376. 71. Olson, K. R.; Gao, Y.; Arif, F.; Arora, K.; Patel, S.; DeLeon, E. R.; Sutton, T. R.; Feelisch, M.; Cortese-Krott, M. M.; Straub, K. D., Metabolism of hydrogen sulfide (H2S) and Production of Reactive Sulfur Species (RSS) by superoxide dismutase. Redox Biol. 2018, 15, 74-85. 72. Miyamoto, R.; Koike, S.; Takano, Y.; Shibuya, N.; Kimura, Y.; Hanaoka, K.; Urano, Y.; Ogasawara, Y.; Kimura, H., Polysulfides (H 2 S n) produced from the interaction of hydrogen sulfide (H 2 S) and nitric oxide (NO) activate TRPA1 channels. Sci. Rep. 2017, 7:45995. 73. Nagy, P.; Winterbourn, C. C., Rapid reaction of hydrogen sulfide with the neutrophil oxidant hypochlorous acid to generate polysulfides. Chem. Res. Toxicol. 2010, 23 (10), 1541-1543. 74. Eberhardt, M.; Dux, M.; Namer, B.; Miljkovic, J.; Cordasic, N.; Will, C.; Kichko, T. I.; De La Roche, J.; Fischer, M.; Suárez, S. A., H 2 S and NO cooperatively regulate vascular tone by activating a neuroendocrine HNO–TRPA1–CGRP signalling pathway. Nature Commun. 2014, 5, 4381. 75. Cortese-Krott, M. M.; Kuhnle, G. G.; Dyson, A.; Fernandez, B. O.; Grman, M.; DuMond, J. F.; Barrow, M. P.; McLeod, G.; Nakagawa, H.; Ondrias, K., Key bioactive reaction products of the NO/H2S interaction are S/N-hybrid species, polysulfides, and nitroxyl. Proc. Natl. Acad. Sci. 2015, 112 (34), E4651-E4660. 76. Xu, Z.; Qiu, Z.; Liu, Q.; Huang, Y.; Li, D.; Shen, X.; Fan, K.; Xi, J.; Gu, Y.; Tang, Y., Converting organosulfur compounds to inorganic polysulfides against resistant bacterial infections. Nat. Commun. 2018, 9, 3713. 77. Nava, M.; Martin-Drumel, M.-A.; Lopez, C. A.; Crabtree, K. N.; Womack, C. C.; Nguyen, T. L.; Thorwirth, S.; Cummins, C. C.; Stanton, J. F.; McCarthy, M. C., Spontaneous and selective formation of HSNO, a crucial intermediate linking H2S and nitroso chemistries. J. Am. Chem. Soc. 2016, 138 (36), 11441-11444. 78. Shen, J.; Walsh, B. J.; Flores-Mireles, A. L.; Peng, H.; Zhang, Y.; Zhang, Y.; Trinidad, J. C.; Hultgren, S. J.; Giedroc, D. P., Hydrogen sulfide sensing through reactive sulfur species (RSS) and nitroxyl (HNO) in Enterococcus faecalis. ACS Chem. Biol. 2018, 13 (6), 1610-1620. 79. Zhang, T.; Ono, K.; Tsutsuki, H.; Ihara, H.; Islam, W.; Akaike, T.; Sawa, T., Enhanced Cellular Polysulfides Negatively Regulate TLR4 Signaling and Mitigate Lethal Endotoxin Shock. Cell Chem. Biol. 2019, 26, 686-698. 80. Kang, J.; Li, Z.; Organ, C. L.; Park, C.-M.; Yang, C.-t.; Pacheco, A.; Wang, D.; Lefer, D. J.; Xian, M., pH-controlled hydrogen sulfide release for myocardial ischemia-reperfusion injury. J. Am. Chem. Soc. 2016, 138 (20), 6336-6339. 81. Yu, B.; Zheng, Y.; Yuan, Z.; Li, S.; Zhu, H.; De La Cruz, L. K.; Zhang, J.; Ji, K.; Wang, S.; Wang, B., Toward direct protein S-persulfidation:


Page 13 of 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a prodrug approach that directly delivers hydrogen persulfide. J. Am. Chem. Soc. 2018, 140 (1), 30-33. 82. Khodade, V. S.; Toscano, J. P., Development of S-Substituted Thioisothioureas as Efficient Hydropersulfide Precursors. J. Am. Chem. Soc. 2018, 140 (50), 17333-17337. 83. Ezeriņa, D.; Takano, Y.; Hanaoka, K.; Urano, Y.; Dick, T. P., Nacetyl cysteine functions as a fast-acting antioxidant by triggering intracellular H2S and sulfane sulfur production. Cell Chem. Biol. 2018, 25 (4), 447-459. 84. Das, T.; Huie, R. E.; Neta, P.; Padmaja, S., Reduction Potential of the Sulfhydryl Radical: Pulse Radiolysis and Laser Flash Photolysis Studies of the Formation and Reactions of⊙ SH and HSSH⊙-in Aqueous Solutions. J. Phys. Chem. A 1999, 103 (27), 5221-5226.

85. Benchoam, D.; Cuevasanta, E.; Möller, M. N.; Alvarez, B., Hydrogen Sulfide and Persulfides Oxidation by Biologically Relevant Oxidizing Species. Antioxidants 2019, 8 (2), 48. 86. Akam, E. A.; Tomat, E., Targeting iron in colon cancer via glycoconjugation of thiosemicarbazone prochelators. Bioconj. Chem. 2016, 27 (8), 1807-1812. 87. Gun, J.; Modestov, A. D.; Kamyshny, A.; Ryzkov, D.; Gitis, V.; Goifman, A.; Lev, O.; Hultsch, V.; Grischek, T.; Worch, E., Electrospray ionization mass spectrometric analysis of aqueous polysulfide solutions. Microchimica Acta 2004, 146 (3-4), 229-237. 88. Ravel, B.; Newville, M., ATHENA, ARTEMIS, HEPHAESTUS: data analysis for X-ray absorption spectroscopy using IFEFFIT. J. Synchrotron Radiat. 2005, 12 (4), 537-541.




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Potent Activation of Indoleamine 2,3-Dioxygenase by Polysulfides

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