Histidine 55 of tryptophan 2,3-dioxygenase is not an active site base but regulates catalysis by controlling substrate binding

Tryptophan 2,3-dioxygenase (TDO) from Xanthomonas campestris is a highly specific heme-containing enzyme from a small family of homologous enzymes, which includes indoleamine 2,3-dioxygenase (IDO). The structure of wild type (WT TDO) in the catalytically active, ferrous (Fe (2+)) form and in complex with its substrate l-tryptophan ( l-Trp) was recently reported [Forouhar et al. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 473-478] and revealed that histidine 55 hydrogen bonds to l-Trp, precisely positioning it in the active site and implicating it as a possible active site base. In this study the substitution of the active site residue histidine 55 by alanine and serine (H55A and H55S) provides insight into the molecular mechanism used by the enzyme to control substrate binding. We report the crystal structure of the H55A and H55S mutant forms at 2.15 and 1.90 A resolution, respectively, in binary complexes with l-Trp. These structural data, in conjunction with potentiometric and kinetic studies on both mutants, reveal that histidine 55 is not essential for turnover but greatly disfavors the mechanistically unproductive binding of l-Trp to the oxidized enzyme allowing control of catalysis. This is demonstrated by the difference in the K d values for l-Trp binding to the two oxidation states of wild-type TDO (3.8 mM oxidized, 4.1 microM reduced), H55A TDO (11.8 microM oxidized, 3.7 microM reduced), and H55S TDO (18.4 microM oxidized, 5.3 microM reduced).     

Synthesis of novel tryptanthrin derivatives as dual inhibitors of indoleamine 2,3-dioxygenase 1 and tryptophan 2,3-dioxygenase

Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO) are promising drug development targets due to their implications in pathologies such as cancer and neurodegenerative diseases. The search for IDO1 inhibitor has been intensely pursued but there is a paucity of potent TDO and IDO1/TDO dual inhibitors. Natural product tryptanthrin has been confirmed to bear IDO1 and/or TDO inhibitory activities. Herein, twelve novel tryptanthrin derivatives were synthesized and evaluated for the IDO1 and TDO inhibitory potency. All of the compounds were found to be IDO1/TDO dual inhibitors, in particular, compound 9a and 9b bore IDO1 inhibitory activity similar to that of INCB024360, and compound 5a and 9b had remarkable TDO inhibitory activity superior to that of the well-known TDO inhibitor LM10. This work enriches the collection of IDO1/TDO dual inhibitors and provides chemical molecules for potential development into drugs.     

A kinetic, spectroscopic, and redox study of human tryptophan 2,3-dioxygenase

The family of heme dioxygenases, as exemplified by indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase, catalyzes the oxidative cleavage of L-tryptophan to N-formylkynurenine. Here, we describe a bacterial expression system for human tryptophan 2,3-dioxygenase (rhTDO) together with spectroscopic, kinetic, and redox analyses. We find unexpected differences between human tryptophan 2,3-dioxygenase and human indoleamine 2,3-dioxygenase [Chauhan et al. (2008) Biochemistry 47, 4761-4769 ]. Thus, in contrast to indoleamine 2,3-dioxygenase, the catalytic ferrous-oxy complex of rhTDO is not observed, nor does the enzyme discriminate against substrate binding to the ferric derivative. In addition, we show that the rhTDO is also catalytically active in the ferric form. These new findings illustrate that significant mechanistic differences exist across the heme dioxygenase family, and the data are discussed within this broader framework.     

Comparative effects of oxygen on indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase of the kynurenine pathway

Indoleamine 2,3-dioxygenase (IDO) reacts with either oxygen or superoxide and tryptophan (trp) or other indoleamines while tryptophan 2,3-dioxygenase (TDO) reacts with oxygen and is specific for trp. These enzymes catalyze the rate-limiting step in the kynurenine (KYN) pathway from trp to quinolinic acid (QA) with TDO in kidney and liver and IDO in many tissues, including brain where it is low but inducible. QA, which does not cross the blood-brain barrier, is an excitotoxin found in the CNS during various pathologies and is associated with convulsions. We proposed that HBO-induced convulsions result from increased flux through the KYN pathway via oxygen stimulation of IDO. To test this, TDO and IDO of liver and brain, respectively, of Sprague Dawley rats were assayed with oxygen from 0 to 6.2 atm HBO. TDO activity was appreciable at even 30 microM oxygen and rose steeply to a maximum at 40 microM. Conversely, IDO had almost no detectable activity at or below 100 microM oxygen and maximum activity was not reached until about 1150 microM. (Plasma contains about 215 microM oxygen and capillaries about 20 microM oxygen when rats breathe air.) KYN was 60% higher in brains of HBO-convulsed rats compared to rats breathing air. While the oxygen concentration inside cells of rats breathing air or HBO is not known precisely, it is clear that the rate-limiting, IDO-catalyzed step in the brain KYN pathway (but not liver TDO) can be greatly accelerated in rats breathing HBO.     

A comprehensive comparison of the metazoan tryptophan degrading enzymes

Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) have an independent origin; however, they have distinctly evolved to catalyze the same reaction. In general, TDO is a single-copy gene in each metazoan species, and TDO enzymes demonstrate similar enzyme activity regardless of their biological origin. In contrast, multiple IDO paralogues are observed in many species, and they display various enzymatic properties. Similar to vertebrate IDO2, invertebrate IDOs generally show low affinity/catalytic efficiency for L-Trp. Meanwhile, two IDO isoforms from scallop (IDO-I and -III) and sponge IDOs show high L-Trp catalytic activity, which is comparable to vertebrate IDO1. Site-directed mutagenesis experiments have revealed that primarily two residues, Tyr located at the 2nd residue on the F-helix (F2^nd) and His located at the 9th residue on the G-helix (G9^th), are crucial for the high affinity/catalytic efficiency of these ‘high performance’ invertebrate IDOs. Conversely, those two amino acid substitutions (F2^nd/Tyr and G9^th/His) resulted in high affinity and catalytic activity in other molluscan ‘low performance’ IDOs. In human IDO1, G9^th is Ser¹⁶⁷, whereas the counterpart residue of G9^th in human TDO is His⁷⁶. Previous studies have shown that Ser¹⁶⁷ could not be substituted by His because the human IDO1 Ser¹⁶⁷His variant showed significantly low catalytic activity. However, this may be specific for human IDO1 because G9^th/His was demonstrated to be very effective in increasing the L-Trp affinity even in vertebrate IDOs. Therefore, these findings indicate that the active sites of TDO and IDO are more similar to each other than previously expected.     

Contributions of tryptophan 2,3-dioxygenase and indoleamine 2,3-dioxygenase to the conversion of d-tryptophan to nicotinamide analyzed by using tryptophan 2,3-dioxygenase-knockout mice

We investigated the contribution percentage of tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) to the conversion of d-tryptophan to nicotinamide in TDO-knockout mice. The calculated percentage conversions indicated that TDO and IDO oxidized 70 and 30%, respectively, of the dietary l-tryptophan. These results indicate that both TDO and IDO biosynthesize nicotinamide from d-tryptophan and l-tryptophan in mice.     

Molecular evolution of bacterial indoleamine 2,3-dioxygenase

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are tryptophan-degrading enzymes that catalyze the first step in L-Trp catabolism via the kynurenine pathway. In mammals, TDO is mainly expressed in the liver and primarily supplies nicotinamide adenine dinucleotide (NAD⁺). TDO is widely distributed from mammals to bacteria. Active IDO enzymes have been reported only in vertebrates and fungi. In mammals, IDO activity plays a significant role in the immune system while in fungal species, IDO is constitutively expressed and supplies NAD⁺, like mammalian TDO. A search of genomic databases reveals that some bacterial species also have a putative IDO gene. A phylogenetic analysis clustered bacterial IDOs into two groups, group I or group II bacterial IDOs. The catalytic efficiencies of group I bacterial IDOs were very low and they are suspected not to contribute significantly to L-Trp metabolism. The bacterial species bearing the group I bacterial IDO are scattered across a few phyla and no phylogenetically close relationship is observed between them. This suggests that the group I bacterial IDOs might be acquired by horizontal gene transmission that occurred in each lineage independently. In contrast, group II bacterial IDOs showed rather high catalytic efficiency. Particularly, the enzymatic characteristics (K_m, V_max and inhibitor selectivity) of the Gemmatimonas aurantiaca IDO are comparable to those of mammalian IDO1, although comparison of the IDO sequences does not suggest a close evolutionary relationship. In several bacteria, TDO and the kynureninase gene (kynU) are clustered on their chromosome suggesting that these genes could be transcribed in an operon. Interestingly, G. aurantiaca has no TDO, and the IDO is clustered with kynU on its chromosome. Although the G. aurantiaca also has NadA and NadB to synthesize a quinolinic acid (a precursor of NAD⁺) via the aspartate pathway, the high activity of the G. aurantiaca IDO flanking the kynU gene suggests its IDO has a function similar to eukaryotic enzymes.     

Induction of tryptophan 2,3-dioxygenase in the mouse endometrium during implantation

Indoleamine 2,3-dioxygenase (IDO) is expressed in trophoblasts and defends the conceptus against rejection by reducing the tryptophan level and suppressing the T cell activity. We isolated a cDNA for tryptophan 2,3-dioxygenase (TDO), another key catabolizing enzyme of tryptophan, from a mouse uterus cDNA library enriched with pregnancy-induced genes. Northern blot and in situ hybridization analyses demonstrated that the TDO mRNA was induced in the decidualized stromal cells around the implanted embryo at the time of implantation. The expression was then upregulated and primarily localized at the mesometrial decidua. TDO mRNA was induced by deciduoma formation as well as embryo transfer but not by ovarian steroid hormones. These findings demonstrated that TDO is induced in the endometrial stromal cells concomitant with decidualization and suggested its involvement in the implantation process by regulating the tryptophan level at the implantation site.     

The first step of the dioxygenation reaction carried out by tryptophan dioxygenase and indoleamine 2,3-dioxygenase as revealed by quantum mechanical/molecular mechanical studies

Tryptophan dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) are two heme-containing enzymes which catalyze the conversion of l-tryptophan to N-formylkynurenine (NFK). In mammals, TDO is mostly expressed in liver and is involved in controlling homeostatic serum tryptophan concentrations, whereas IDO is ubiquitous and is involved in modulating immune responses. Previous studies suggested that the first step of the dioxygenase reaction involves the deprotonation of the indoleamine group of the substrate by an evolutionarily conserved distal histidine residue in TDO and the heme-bound dioxygen in IDO. Here, we used classical molecular dynamics and hybrid quantum mechanical/molecular mechanical methods to evaluate the base-catalyzed mechanism. Our data suggest that the deprotonation of the indoleamine group of the substrate by either histidine in TDO or heme-bound dioxygen in IDO is not energetically favorable. Instead, the dioxygenase reaction can be initiated by a direct attack of heme-bound dioxygen on the C₂=C₃ bond of the indole ring, leading to a protein-stabilized 2,3-alkylperoxide transition state and a ferryl epoxide intermediate, which subsequently recombine to generate NFK. The novel sequential two-step oxygen addition mechanism is fully supported by our recent resonance Raman data that allowed identification of the ferryl intermediate (Lewis-Ballester et al. in Proc Natl Acad Sci USA 106:17371–17376, 2009). The results reveal the subtle differences between the TDO and IDO reactions and highlight the importance of protein matrix in modulating stereoelectronic factors for oxygen activation and the stabilization of both transition and intermediate states.     

Indoleamine 2,3-dioxygenase 2 (IDO2) and the kynurenine pathway: characteristics and potential roles in health and disease

The kynurenine pathway is the major route for the oxidative degradation of the amino acid tryptophan. Activity of the pathway is involved in several disease conditions, both in the periphery and the central nervous system, including cancer, inflammatory disorders, neurological conditions, psychiatric disorders and neurodegenerative diseases. Three enzymes are now known to catalyze the first and rate-limiting step in the catabolism of tryptophan along this pathway: tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO, subsequently named IDO1), both of which have been extensively studied, and a third enzyme, indoleamine 2,3-dioxygenase 2 (IDO2), a relative newcomer to the kynurenine pathway field. The adjuvant chemotherapeutic agent, 1-methyl-d-tryptophan, was intially suggested to target IDO2, implying involvement of IDO2 in tumorigenesis. Subsequently this compound has been suggested to have alternative actions and the physiological and pathophysiological roles of IDO2 are unclear. Targeted genetic interventions and selective inhibitors provide approaches for investigating the biology of IDO2. This review focuses on the current knowledge of IDO2 biology and discusses tools that will assist in further characterizing the enzymes of the kynurenine pathway.     

Indoleamine 2,3-dioxygenase inhibitory activity of derivatives of marine alkaloid tsitsikammamine A

Tsitsikammamines are marine alkaloids whose structure is based on the pyrroloiminoquinone scaffold. These and related compounds have attracted attention due to various interesting biological properties, including cytotoxicity, topoisomerase inhibition, antimicrobial, antifungal and antimalarial activity.Indoleamine 2,3-dioxygenase (IDO1) is a well-established therapeutic target as an important factor in the tumor immune evasion mechanism. In this preliminary communication, we report the inhibitory activity of tsitsikammamine derivatives against IDO1. Tsitsikammamine A analogue 11b displays submicromolar potency in an enzymatic assay. A number of derivatives are also active in a cellular assay while showing little or no activity towards tryptophan 2,3-dioxygenase (TDO), a functionally related enzyme. This IDO1 inhibitory activity is rationalized by molecular modeling studies. An interest is thus established in this class of compounds as a potential source of lead compounds for the development of new pharmaceutically useful IDO1 inhibitors.     

Crystal structure and mechanism of tryptophan 2,3-dioxygenase, a heme enzyme involved in tryptophan catabolism and in quinolinate biosynthesis

The structure of tryptophan 2,3-dioxygenase (TDO) from Ralstonia metallidurans was determined at 2.4 A. TDO catalyzes the irreversible oxidation of l-tryptophan to N-formyl kynurenine, which is the initial step in tryptophan catabolism. TDO is a heme-containing enzyme and is highly specific for its substrate l-tryptophan. The structure is a tetramer with a heme cofactor bound at each active site. The monomeric fold, as well as the heme binding site, is similar to that of the large domain of indoleamine 2,3-dioxygenase, an enzyme that catalyzes the same reaction except with a broader substrate tolerance. Modeling of the putative (S)-tryptophan hydroperoxide intermediate into the active site, as well as substrate analogue and mutagenesis studies, are consistent with a Criegee mechanism for the reaction.     

Redox and spectroscopic properties of human indoleamine 2,3-dioxygenase and a His303Ala variant: implications for catalysis

Indoleamine 2,3-dioxygenase is an important mammalian target that catalyses the oxidative cleavage of l-tryptophan to N-formylkynurenine. In this work, the redox properties of recombinant human indoleamine 2,3-dioxygenase (rhIDO) and its H303A variant have been examined for the first time and the spectroscopic and substrate-binding properties of rhIDO and H303A in the presence and absence of substrate are reported. The Fe(3+)/Fe(2+) reduction potential of H303A was found to be -30 +/- 4 mV; in the presence of l-Trp, this value increases to +16 +/- 3 mV. A variety of spectroscopies indicate that ferric rhIDO at pH 6.6 exists as a mixture of six-coordinate, high-spin, water-bound heme and a low-spin species that contains a second nitrogenous ligand; parallel experiments on H303A are consistent either with His303 as the sixth ligand or with His303 linked to a conformational change that affects this transition. There is an increase in the low-spin component at alkaline pH for rhIDO, but this is not due to hydroxide-bound heme. Substrate binding induces a conformational rearrangement and formation of low-spin, hydroxide-bound heme; analysis of the H303A variant indicates that His303 is not required for this conversion and is not essential for substrate binding. The Fe(3+)/Fe(2+) reduction potential of H303A variant is approximately 70 mV lower than that of rhIDO, leading to a destabilization of the ferrous-oxy complex, which is an obligate intermediate in the catalytic process. In comparison with the properties of other heme enzymes, the data can be used to build a more detailed picture of substrate binding and catalysis in indoleamine 2,3-dioxygenase. The wider implications of these results are discussed in the context of our current understanding of the catalytic mechanism of the enzyme.     

Evolution of Vertebrate Indoleamine 2,3-Dioxygenases

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are tryptophan-degrading enzymes that catalyze the same reaction, the first step in tryptophan catabolism via the kynurenine pathway. TDO is widely distributed among life-forms, being found not only in eukaryotes but also in bacteria. In contrast, IDO has been found only in mammals and yeast to date. However, recent genome and EST projects have identified IDO homologues in non-mammals and found an IDO paralogue that is expressed in mice. In this study, we cloned the frog and fish IDO homologues and the mouse IDO paralogue, and characterized their enzymatic properties using recombinants. The IDOs of lower vertebrates and the mouse IDO paralogue had IDO activity but had 500–1000 times higher K _m values and very low enzyme efficiency compared with mammalian IDOs. It appears that L-Trp is not a true substrate for these enzymes in vivo, although their actual function is unknown. On the phylogenetic tree, these low-activity IDOs, which we have named “proto-IDOs,” formed a cluster that was distinct from the mammalian IDO cluster. The IDO and proto-IDO genes are present tandemly on the chromosomes of mammals, including the marsupial opossum, whereas only the proto-IDO gene is observed in chicken and fish genomes. These results suggest that (mammalian) IDOs arose from proto-IDOs by gene duplication that occurred before the divergence of marsupial and eutherian (placental) mammals in mammalian evolutionary history.     

Antitumour agents as inhibitors of tryptophan 2,3-dioxygenase

The involvement of tryptophan 2,3-dioxygenase (TDO) in cancer biology has recently been described, with the enzyme playing an immunomodulatory role, suppressing antitumour immune responses and promoting tumour cell survival and proliferation. This finding reinforces the need for specific inhibitors of TDO that may potentially be developed for therapeutic use. In this work we have screened ∼2800 compounds from the library of the National Cancer Institute USA and identified seven potent inhibitors of TDO with inhibition constants in the nanomolar or low micromolar range. All seven have antitumour properties, killing various cancer cell lines. For comparison, the inhibition potencies of these compounds were tested against IDO and their inhibition constants are reported. Interestingly, this work reveals that NSC 36398 (dihydroquercetin, taxifolin), with an in vitro inhibition constant of ∼16 μM, is the first TDO-selective inhibitor reported.     

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

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

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

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

Biochemical mechanisms leading to tryptophan 2,3-dioxygenase activation

Tryptophan 2,3-dioxygenase (TDO) is the first enzyme in the tryptophan oxidation pathway. It is a hemoprotein and its heme prosthetic group is present as a heme-ferric (heme-Fe(3+)) form that is not active. To be able to oxidize tryptophan, the heme-Fe(3+) form of the enzyme must be reduced to a heme-ferrous (heme-Fe(2+)) form and this study describes conditions that promote TDO activation. TDO is progressively activated upon mixing with tryptophan in a neutral buffer, which leads to an impression that tryptophan is responsible for TDO activation. Through extensive analysis of factors resulting in TDO activation during incubation with tryptophan, we conclude that tryptophan indirectly activates TDO through promoting the production of reactive oxygen species. This consideration is supported by the virtual elimination of the initial lag phase when either pre-incubated tryptophan solution was used as the substrate or a low concentration of superoxide or hydrogen peroxide was incorporated into the freshly tryptophan and TDO mixture. However, accumulation of these reactive oxygen species also leads to the inactivation of TDO, so that both TDO activation and inactivation proceed with the specific outcome depending greatly on the concentrations of superoxide and hydrogen peroxide. As a consequence, the rate of TDO catalysis varies depending upon the proportion of the active to inactive forms of the enzyme, which is in a dynamic relationship in the reaction mixture. These data provide some insight towards elucidating the molecular regulation of TDO in vivo.     

17O]Water and nitric oxide binding by protocatechuate 4,5-dioxygenase and catechol 2,3-dioxygenase. Evidence for binding of exogenous ligands to the active site Fe2+ of extradiol dioxygenases

Pseudomonas testosteroni protocatechuate 4,5-dioxygenase and Pseudomonas putida catechol 2,3-dioxygenase (metapyrocatechase) catalyze extradiol-type oxygenolytic cleavage of the aromatic ring of their substrates. The essential active site Fe2+ of each enzyme binds nitric oxide (NO) to produce an EPR active complex with an electronic spin of S = 3/2. Hyperfine broadening of the EPR resonances of the nitrosyl complexes by 17O-enriched H2O shows that water is bound directly to the Fe2+ in the native enzymes, but is apparently displaced in substrate complexes. NO is not displaced by either substrates or inhibitors. The EPR spectra of several enzyme-inhibitor-NO complexes are different from those of enzyme-NO or enzyme-substrate-NO complexes and are found to be broadened by 17O-enriched water. The data show that at least 2 and perhaps 3 sites in the Fe ligation can be occupied by exogenous ligands. Furthermore, it is likely that substrates and inhibitors displace water by binding either at or near to the Fe in the nitrosyl complex. Nitric oxide binding is found to be substrate-dependent for each enzyme. Native catechol 2,3-dioxygenase exhibits KD values of 190 microM and 2.0 mM for NO binding in two types of independent sites. Only one type of site is observed in the catechol complex which exhibits a KD for NO of 3.4 microM. One type of NO binding site is observed for both the native and substrate complexed protocatechuate 4,5-dioxygenase with KD values of 360 and 3 microM, respectively. The presence of a specific site in the Fe coordination for NO which is modified in the substrate complex, suggests that O2 binding by the extradiol dioxygenases may also occur at the Fe.