Harnessing solid-state ion exchange for the environmentally benign synthesis of high-efficiency Mn4+-doped phosphors.

In this study, a solid-state ion exchange (SSIE) method is proposed to synthesize a series of Mn4+-doped fluoride phosphors, which avoids the use of HF solution during the Mn4+-doping process. The obtained Mn4+-doped fluoride phosphors exhibit strong red emission with a quantum yield of 46%.

Retinoic acid receptor α activity in proximal tubules prevents kidney injury and fibrosis.

Chronic kidney disease (CKD) is characterized by a gradual loss of kidney function and affects ~13.4% of the global population. Progressive tubulointerstitial fibrosis, driven in part by proximal tubule (PT) damage, is a hallmark of late stages of CKD and contributes to the development of kidney failure, for which there are limited treatment options. Normal kidney development requires signaling by vitamin A (retinol), which is metabolized to retinoic acid (RA), an endogenous agonist for the RA receptors (RARα, ß, γ). RARα levels are decreased in a mouse model of diabetic nephropathy and restored with RA administration; additionally, RA treatment reduced fibrosis. We developed a mouse model in which a spatiotemporal (tamoxifen-inducible) deletion of RARα in kidney PT cells of adult mice causes mitochondrial dysfunction, massive PT injury, and apoptosis without the use of additional nephrotoxic substances. Long-term effects (3 to 4.5 mo) of RARα deletion include increased PT secretion of transforming growth factor ß1, inflammation, interstitial fibrosis, and decreased kidney function, all of which are major features of human CKD. Therefore, RARα's actions in PTs are crucial for PT homeostasis, and loss of RARα causes injury and a key CKD phenotype.

A retinoic acid receptor ß2 agonist attenuates transcriptome and metabolome changes underlying nonalcohol-associated fatty liver disease.

Nonalcohol-associated fatty liver disease (NAFLD) is characterized by excessive hepatic accumulation of fat that can progress to steatohepatitis, and currently, therapeutic options are limited. Using a high-fat diet (HFD) mouse model of NAFLD, we determined the effects of the synthetic retinoid, AC261066, a selective retinoic acid receptor ß2 (RARß2) agonist, on the global liver transcriptomes and metabolomes of mice with dietary-induced obesity (DIO) using genome-wide RNA-seq and untargeted metabolomics. We found that AC261066 limits mRNA increases in several presumptive NAFLD driver genes, including Pklr, Fasn, Thrsp, and Chchd6. Importantly, AC261066 limits the increases in the transcript and protein levels of KHK, a key enzyme for fructose metabolism, and causes multiple changes in liver metabolites involved in fructose metabolism. In addition, in cultured murine hepatocytes, where exposure to fructose and palmitate results in a profound increase in lipid accumulation, AC261066 limits this lipid accumulation. Importantly, we demonstrate that in a human hepatocyte cell line, RARß is required for the inhibitory effects of AC261066 on palmitate-induced lipid accumulation. Finally, our data indicate that AC261066 inhibits molecular events underpinning fibrosis and exhibits anti-inflammatory effects. In conclusion, changes in the transcriptome and metabolome indicate that AC261066 affects molecular changes underlying multiple aspects of NAFLD, including steatosis and fibrosis. Therefore, we suggest that AC261066 may have potential as an effective therapy for NAFLD.

ETHE1 overexpression promotes SIRT1 and PGC1α mediated aerobic glycolysis, oxidative phosphorylation, mitochondrial biogenesis and colorectal cancer.

Ethylmalonic Encephalopathy Protein 1 (ETHE1) is a sulfur dioxygenase that regulates cellular H2S levels. We previously demonstrated a significant increase of ETHE1 expression in "single-hit" colon epithelial cells from crypts of patients with Familial Adenomatous Polyposis (FAP). Here, we report elevated levels of ETHE1 expression and increased mitochondrial density occurring in-situ in phenotypically normal FAP colorectal mucosa. We also found that constitutive expression of ETHE1 increased aerobic glycolysis ("Warburg effect"), oxidative phosphorylation, and mitochondrial biogenesis in colorectal cancer (CRC) cell lines, thereby depleting H2S which relieved the inhibition of phosphodiesterase (PDE), and increased adenosine monophosphate (AMP) levels. This led to activation of the energy sensing AMP-activated protein kinase (AMPKp), Sirtuin1 (SIRT1) and peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), a master regulator of mitochondrial biogenesis. By contrast, shRNA silencing of ETHE1 reduced PDE activity, AMPKp/SIRT1/PGC1α levels and mitochondrial biogenesis. Constitutive expression of ETHE1 accelerated both CRC cell xenograft and orthotopic patient derived xenograft CRC cell growth in vivo. Overall, our data nominate elevated ETHE1 expression levels as a novel biomarker and potential therapeutic target for the prevention of CRC tumorigenesis.

Different Effects of Knockouts in ALDH2 and ACSS2 on Embryonic Stem Cell Differentiation.

BACKGROUND: Ethanol (EtOH) is a teratogen that causes severe birth defects, but the mechanisms by which EtOH affects stem cell differentiation are unclear. Our goal here is to examine the effects of EtOH and its metabolites, acetaldehyde (AcH) and acetate, on embryonic stem cell (ESC) differentiation. METHODS: We designed ESC lines in which aldehyde dehydrogenase (ALDH2, NCBI#11669) and acyl-CoA synthetase short-chain family member 2 (ACSS2, NCBI#60525) were knocked out by CRISPR-Cas9 technology. We selected these genes because of their key roles in EtOH oxidation in order to dissect the effects of EtOH metabolism on differentiation. RESULTS: By using kinetic assays, we confirmed that AcH is primarily oxidized by ALDH2 rather than ALDH1A2. We found increases in mRNAs of differentiation-associated genes (Hoxa1, Cyp26a1, and RARß2) upon EtOH treatment of WT and Acss2-/- ESCs, but not Aldh2-/- ESCs. The absence of ALDH2 reduced mRNAs of some pluripotency factors (Nanog, Sox2, and Klf4). Treatment of WT ESCs with AcH or 4-hydroxynonenal (4-HNE), another substrate of ALDH2, increased differentiation-associated transcripts compared to levels in untreated cells. mRNAs of genes involved in retinoic acid (RA) synthesis (Stra6 and Rdh10) were also increased by EtOH, AcH, and 4-HNE treatment. Retinoic acid receptor-γ (RARγ) is required for both EtOH- and AcH-mediated increases in Hoxa1 and Stra6, demonstrating the critical role of RA:RARγ signaling in AcH-induced ESC differentiation. CONCLUSIONS: ACSS2 knockouts showed no changes in differentiation phenotype, while pluripotency-related transcripts were decreased in ALDH2 knockout ESCs. We demonstrate that AcH increases differentiation-associated mRNAs in ESCs via RARγ.

High-fructose corn syrup enhances intestinal tumor growth in mice.

Excessive consumption of beverages sweetened with high-fructose corn syrup (HFCS) is associated with obesity and with an increased risk of colorectal cancer. Whether HFCS contributes directly to tumorigenesis is unclear. We investigated the effects of daily oral administration of HFCS in adenomatous polyposis coli (APC) mutant mice, which are predisposed to develop intestinal tumors. The HFCS-treated mice showed a substantial increase in tumor size and tumor grade in the absence of obesity and metabolic syndrome. HFCS increased the concentrations of fructose and glucose in the intestinal lumen and serum, respectively, and the tumors transported both sugars. Within the tumors, fructose was converted to fructose-1-phosphate, leading to activation of glycolysis and increased synthesis of fatty acids that support tumor growth. These mouse studies support the hypothesis that the combination of dietary glucose and fructose, even at a moderate dose, can enhance tumorigenesis.

In-situ XRD and EDS method study on the oxidation behaviour of Ni-Cu sulphide ore.

The oxidation mechanism of sulfides is the key issue during the sulphide-metallurgy process. In this study, the phase transformation and element migration were clearly demonstrated by in-situ laboratory-based X-ray diffraction (XRD) and energy-dispersive X-ray spectroscopy (EDS), respectively. The reaction sequence and a four-step oxidation mechanism were proposed and identified. The elemental distribution demonstrated that at a low temperature, the Fe atoms diffused outward and the Ni/Cu atoms migrated toward the inner core, whereas the opposite diffusion processes were observed at a higher temperature. Importantly, the unique visual presentation of the oxidation behaviour provided by the combination of in-situ XRD and EDS might be useful for optimising the process parameters to improve the Ni/Cu extraction efficiency during Ni-Cu sulphide metallurgy.

Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH.

More than half of human colorectal cancers (CRCs) carry either KRAS or BRAF mutations and are often refractory to approved targeted therapies. We found that cultured human CRC cells harboring KRAS or BRAF mutations are selectively killed when exposed to high levels of vitamin C. This effect is due to increased uptake of the oxidized form of vitamin C, dehydroascorbate (DHA), via the GLUT1 glucose transporter. Increased DHA uptake causes oxidative stress as intracellular DHA is reduced to vitamin C, depleting glutathione. Thus, reactive oxygen species accumulate and inactivate glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Inhibition of GAPDH in highly glycolytic KRAS or BRAF mutant cells leads to an energetic crisis and cell death not seen in KRAS and BRAF wild-type cells. High-dose vitamin C impairs tumor growth in Apc/Kras(G12D) mutant mice. These results provide a mechanistic rationale for exploring the therapeutic use of vitamin C for CRCs with KRAS or BRAF mutations.

S-sulfhydration/desulfhydration and S-nitrosylation/denitrosylation: a common paradigm for gasotransmitter signaling by H2S and NO.

Sulfhydryl groups on protein Cys residues undergo an array of oxidative reactions and modifications, giving rise to a virtual redox zip code with physiological and pathophysiological relevance for modulation of protein structure and functions. While over two decades of studies have established NO-dependent S-nitrosylation as ubiquitous and fundamental for the regulation of diverse protein activities, proteomic methods for studying H2S-dependent S-sulfhydration have only recently been described and now suggest that this is also an abundant modification with potential for global physiological importance. Notably, protein S-sulfhydration and S-nitrosylation bear striking similarities in terms of their chemical and biological determinants, as well as reversal of these modifications via group-transfer to glutathione, followed by the removal from glutathione by enzymes that have apparently evolved to selectively catalyze denitrosylation and desulfhydration. Here we review determinants of protein and low-molecular-weight thiol S-sulfhydration/desulfhydration, similarities with S-nitrosylation/denitrosylation, and methods that are being employed to investigate and quantify these gasotransmitter-mediated cell signaling systems.

Regulatory role of Glu546 in flavin mononucleotide-heme electron transfer in human inducible nitric oxide synthase.

Nitric oxide (NO) production by mammalian NO synthase (NOS) is believed to be regulated by the docking of the flavin mononucleotide (FMN) domain in one subunit of the dimer onto the heme domain of the adjacent subunit. Glu546, a conserved charged surface residue of the FMN domain in human inducible NOS (iNOS), is proposed to participate in the interdomain FMN/heme interactions [Sempombe et al. Inorg. Chem.2011, 50, 6869-6861]. In the present work, we further investigated the role of the E546 residue in the FMN-heme interdomain electron transfer (IET), a catalytically essential step in the NOS enzymes. Laser flash photolysis was employed to directly measure the FMN-heme IET kinetics for the E546N mutant of human iNOS oxygenase/FMN (oxyFMN) construct. The temperature dependence of the IET kinetics was also measured over the temperature range of 283-304 K to determine changes in the IET activation parameters. The E546N mutation was found to retard the IET by significantly raising the activation entropic barrier. Moreover, pulsed electron paramagnetic resonance data showed that the geometry of the docked FMN/heme complex in the mutant is basically the same as in the wild type construct, whereas the probability of formation of such a complex is about twice lower. These results indicate that the retarded IET in the E546N mutant is not caused by an altered conformation of the docked FMN/heme complex, but by a lower population of the IET-active conformation. In addition, the negative activation entropy of the mutant is still substantially lower than that of the holoenzyme. This supports a mechanism by which the FMN domain can modify the IET through altering probability of the docked state formation.

Catalytic intermediates of inducible nitric-oxide synthase stabilized by the W188H mutation.

Nitric-oxide synthase (NOS) catalyzes nitric oxide (NO) synthesis via a two-step process: L-arginine (L-Arg) → N-hydroxy-L-arginine → citrulline + NO. In the active site the heme is coordinated by a thiolate ligand, which accepts a H-bond from a nearby tryptophan residue, Trp-188. Mutation of Trp-188 to histidine in murine inducible NOS was shown to retard NO synthesis and allow for transient accumulation of a new intermediate with a Soret maximum at 420 nm during the L-Arg hydroxylation reaction (Tejero, J., Biswas, A., Wang, Z. Q., Page, R. C., Haque, M. M., Hemann, C., Zweier, J. L., Misra, S., and Stuehr, D. J. (2008) J. Biol. Chem. 283, 33498-33507). However, crystallographic data showed that the mutation did not perturb the overall structure of the enzyme. To understand how the proximal mutation affects the oxygen chemistry, we carried out biophysical studies of the W188H mutant. Our stopped-flow data showed that the 420-nm intermediate was not only populated during the L-Arg reaction but also during the N-hydroxy-L-arginine reaction. Spectroscopic data and structural analysis demonstrated that the 420-nm intermediate is a hydroxide-bound ferric heme species that is stabilized by an out-of-plane distortion of the heme macrocycle and a cation radical centered on the tetrahydrobiopterin cofactor. The current data add important new insights into the previously proposed catalytic mechanism of NOS (Li, D., Kabir, M., Stuehr, D. J., Rousseau, D. L., and Yeh, S. R. (2007) J. Am. Chem. Soc. 129, 6943-6951).

An unconventional hexacoordinated flavohemoglobin from Mycobacterium tuberculosis.

Being an obligate aerobe, Mycobacterium tuberculosis faces a number of energetic challenges when it encounters hypoxia and environmental stress during intracellular infection. Consequently, it has evolved innovative strategies to cope with these unfavorable conditions. Here, we report a novel flavohemoglobin (MtbFHb) from M. tuberculosis that exhibits unique features within its heme and reductase domains distinct from conventional FHbs, including the absence of the characteristic hydrogen bonding interactions within the proximal heme pocket and mutations in the FAD and NADH binding regions of the reductase domain. In contrast to conventional FHbs, it has a hexacoordinate low-spin heme with a proximal histidine ligand lacking imidazolate character and a distal heme pocket with a relatively low electrostatic potential. Additionally, MtbFHb carries a new FAD binding site in its reductase domain similar to that of D-lactate dehydrogenase (D-LDH). When overexpressed in Escherichia coli or Mycobacterium smegmatis, MtbFHb remained associated with the cell membrane and exhibited D-lactate:phenazine methosulfate reductase activity and oxidized D-lactate into pyruvate by converting the heme iron from Fe(3+) to Fe(2+) in a FAD-dependent manner, indicating electron transfer from D-lactate to the heme via FAD cofactor. Under oxidative stress, MtbFHb-expressing cells exhibited growth advantage with reduced levels of lipid peroxidation. Given the fact that D-lactate is a byproduct of lipid peroxidation and that M. tuberculosis lacks the gene encoding D-LDH, we propose that the novel D-lactate metabolizing activity of MtbFHb uniquely equips M. tuberculosis to balance the stress level by protecting the cell membrane from oxidative damage via cycling between the Fe(3+)/Fe(2+) redox states.

Role of the distal hydrogen-bonding network in regulating oxygen affinity in the truncated hemoglobin III from Campylobacter jejuni.

Oxygen affinity in heme-containing proteins is determined by a number of factors, such as the nature and conformation of the distal residues that stabilize the heme bound-oxygen via hydrogen-bonding interactions. The truncated hemoglobin III from Campylobacter jejuni (Ctb) contains three potential hydrogen-bond donors in the distal site: TyrB10, TrpG8, and HisE7. Previous studies suggested that Ctb exhibits an extremely slow oxygen dissociation rate due to an interlaced hydrogen-bonding network involving the three distal residues. Here we have studied the structural and kinetic properties of the G8(WF) mutant of Ctb and employed state-of-the-art computer simulation methods to investigate the properties of the O(2) adduct of the G8(WF) mutant, with respect to those of the wild-type protein and the previously studied E7(HL) and/or B10(YF) mutants. Our data indicate that the unique oxygen binding properties of Ctb are determined by the interplay of hydrogen-bonding interactions between the heme-bound ligand and the surrounding TyrB10, TrpG8, and HisE7 residues.

Ferryl derivatives of human indoleamine 2,3-dioxygenase.

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

Novel flavohemoglobins of mycobacteria.

Flavohemoglobins (flavoHbs) constitute a distinct class of chimeric hemoglobins in which a globin domain is coupled with a ferredoxin reductase such as FAD- and NADH-binding modules. Structural features and active site of heme and reductase domains are highly conserved in various flavoHbs. A new class of flavoHbs, displaying crucial differences in functionally conserved regions of heme and reductase domains, have been identified in mycobacteria. Mining of microbial genome data indicated that the occurrence of such flavoHbs might be restricted to a small group of microbes unlike conventional flavoHbs that are widespread among prokaryotes and lower eukaryotes. One of the representative flavoHbs of this class, encoded by Rv0385 gene (MtbFHb) of Mycobacterium tuberculosis, has been cloned, expressed, and characterized. The ferric and deoxy spectra of MtbFHb displayed a hexacoordinate state indicating that its distal site may be occupied by an intrinsic amino acid or an external ligand and it may not be involved in nitric oxide detoxification. Phylogenetic analysis revealed that mycobacterial flavoHbs constitute a separate cluster distinct from conventional flavoHbs and may have novel function(s).

Ligand migration in human indoleamine-2,3 dioxygenase.

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

Spectroscopic studies of ligand and substrate binding to human indoleamine 2,3-dioxygenase.

Human indoleamine 2,3-dioxygenase (hIDO) is an intracellular heme-containing enzyme, which catalyzes the initial and rate-determining step of l-tryptophan (l-Trp) metabolism via the kynurenine pathway in nonhepatic tissues. Steady-state kinetic data showed that hIDO exhibits substrate inhibition behavior, implying the existence of a second substrate binding site in the enzyme, although so far there is no direct evidence supporting it. The kinetic data also revealed that the K(m) of l-Trp (15 microM) is approximately 27-fold lower than the K(d) of l-Trp (0.4 mM) for the ligand-free ferrous enzyme, suggesting that O(2) binding proceeds l-Trp binding during the catalytic cycle. With cyanide as a structural probe, we have investigated the thermodynamic and kinetic parameters associated with ligand and substrate binding to hIDO. Equilibrium titration studies show that the cyanide adduct is capable of binding two l-Trp molecules, with K(d) values of 18 microM and 26 mM. The data offer the first direct evidence of the second substrate binding site in hIDO. Kinetic studies demonstrate that prebinding of l-Trp to the enzyme retards cyanide binding by approximately 13-fold, while prebinding of cyanide to the enzyme facilitates l-Trp binding by approximately 22-fold. The data support the view that during the active turnover of the enzyme it is kinetically more favored to bind O(2) prior to l-Trp.

The single-domain globin from the pathogenic bacterium Campylobacter jejuni: novel D-helix conformation, proximal hydrogen bonding that influences ligand binding, and peroxidase-like redox properties.

The food-borne pathogen Campylobacter jejuni possesses a single-domain globin (Cgb) whose role in detoxifying nitric oxide has been unequivocally demonstrated through genetic and molecular approaches. The x-ray structure of cyanide-bound Cgb has been solved to a resolution of 1.35 A. The overall fold is a classic three-on-three alpha-helical globin fold, similar to that of myoglobin and Vgb from Vitreoscilla stercoraria. However, the D region (defined according to the standard globin fold nomenclature) of Cgb adopts a highly ordered alpha-helical conformation unlike any previously characterized members of this globin family, and the GlnE7 residue has an unexpected role in modulating the interaction between the ligand and the TyrB10 residue. The proximal hydrogen bonding network in Cgb demonstrates that the heme cofactor is ligated by an imidazolate, a characteristic of peroxidase-like proteins. Mutation of either proximal hydrogen-bonding residue (GluH23 or TyrG5) results in the loss of the high frequency nu(Fe-His) stretching mode (251 cm(-1)), indicating that both residues are important for maintaining the anionic character of the proximal histidine ligand. Cyanide binding kinetics for these proximal mutants demonstrate for the first time that proximal hydrogen bonding in globins can modulate ligand binding kinetics at the distal site. A low redox midpoint for the ferrous/ferric couple (-134 mV versus normal hydrogen electrode at pH 7) is consistent with the peroxidase-like character of the Cgb active site. These data provide a new insight into the mechanism via which Campylobacter may survive host-derived nitrosative stress.

Role of copper ion in regulating ligand binding in a myoglobin-based cytochrome C oxidase model.

Cytochrome c oxidase (CcO), the terminal enzyme in the mitochondrial respiratory chain, catalyzes the four-electron reduction of dioxygen to water in a binuclear center comprised of a high-spin heme (heme a(3)) and a copper atom (Cu(B)) coordinated by three histidine residues. As a minimum model for CcO, a mutant of sperm whale myoglobin, named Cu(B)Mb, has been engineered, in which a copper atom is held in the distal heme pocket by the native E7 histidine and two nonnative histidine residues. In this work, the role of the copper in regulating ligand binding in Cu(B)Mb was investigated. Resonance Raman studies show that the presence of copper in CO-bound Cu(B)Mb leads to a CcO-like distal heme pocket. Stopped-flow data show that, upon the initiation of the CO binding reaction, the ligand first binds to the Cu(+); it subsequently transfers from Cu(+) to Fe(2+) in an intramolecular process, similar to that reported for CcO. The high CO affinity toward Cu(+) and the slow intramolecular CO transfer rate between Cu(+) and Fe(2+) in the Cu(B)Mb/Cu(+) complex are analogous to those in Thermus thermophilus CcO (TtCcO) but distinct from those in bovine CcO (bCcO). Additional kinetic studies show that, upon photolysis of the NO-bound Cu(B)Mb/Cu(+) complex, the photolyzed ligand transiently binds to Cu(+) and subsequently rebinds to Fe(2+), accounting for the 100% geminate recombination yield, similar to that found in TtCcO. The data demonstrate that the Cu(B)Mb/Cu(+) complex reproduces essential structural and kinetic features of CcO and that the complex is more akin to TtCcO than to bCcO.

Evidence for a ferryl intermediate in a heme-based dioxygenase.

In contrast to the wide spectrum of cytochrome P450 monooxygenases, there are only 2 heme-based dioxygenases in humans: tryptophan dioxygenase (hTDO) and indoleamine 2,3-dioxygenase (hIDO). hTDO and hIDO catalyze the same oxidative ring cleavage reaction of L-tryptophan to N-formyl kynurenine, the initial and rate-limiting step of the kynurenine pathway. Despite immense interest, the mechanism by which the 2 enzymes execute the dioxygenase reaction remains elusive. Here, we report experimental evidence for a key ferryl intermediate of hIDO that supports a mechanism in which the 2 atoms of dioxygen are inserted into the substrate via a consecutive 2-step reaction. This finding introduces a paradigm shift in our understanding of the heme-based dioxygenase chemistry, which was previously believed to proceed via simultaneous incorporation of both atoms of dioxygen into the substrate. The ferryl intermediate is not observable during the hTDO reaction, highlighting the structural differences between the 2 dioxygenases, as well as the importance of stereoelectronic factors in modulating the reactions.