Human indoleamine 2,3-dioxygenase (IDO) catalyzes the cleavage of the pyrrol ring of L-Trp and incorporates both atoms of a molecule of oxygen (O 2). Here we report on the x-ray crystal structure of human IDO, complexed with the ligand inhibitor 4-phenylimidazole and cyanide. The overall structure of IDO shows two ␣-helical domains with the heme between them. A264 of the flexible loop in the heme distal side is in close proximity to the iron. A mutant analysis shows that none of the polar amino acid residues in the distal heme pocket are essential for activity, suggesting that, unlike the heme-containing monooxygenases (i.e., peroxidase and cytochrome P450), no protein group of IDO is essential in dioxygen activation or proton abstraction. These characteristics of the IDO structure provide support for a reaction mechanism involving the abstraction of a proton from the substrate by iron-bound dioxygen. Inactive mutants (F226A, F227A, and R231A) retain substratebinding affinity, and an electron density map reveals that 2-(Ncyclohexylamino)ethane sulfonic acid is bound to these residues, mimicking the substrate. These findings suggest that strict shape complementarities between the indole ring of the substrate and the protein side chains are required, not for binding, but, rather, to permit the interaction between the substrate and iron-bound dioxygen in the first step of the reaction. This study provides the structural basis for a heme-containing dioxygenase mechanism, a missing piece in our understanding of heme chemistry.iron ͉ kynurenine ͉ tryptophan ͉ x-ray crystallography O xygenases (1) are metal-containing enzymes that catalyze the incorporation of a molecule of oxygen (O 2 ) into the substrate, and thus play a crucial role in the metabolism and synthesis of a variety of biological substances. Two types of oxygenase are currently known: monooxygenases (scheme I) and dioxygenases (scheme II):In the 1950s and 1960s, Hayaishi and coworkers reported that two heme-containing dioxygenases, indoleamine 2,3-dioxygenase (IDO) (2) and tryptophan 2,3-dioxygenase (TDO) (3), catalyze the initial and rate-limiting step of L-Trp catabolism in the kynurenine (Kyn) pathway (4). This step involves the oxidative cleavage of the 2,3 double bond in the indole moiety of L-Trp, resulting in the production of N-formyl Kyn. Increased levels of the Kyn pathway metabolites quinolinic acid and 3-hydroxykynurenine (3OHKyn) have been observed in a number of neurological or psychiatric disorders. L-Trp-derived UV filters (Kyn and 3OHKyn glucoside) can bind to the lens protein and appear to be mainly responsible for the nuclear cataract (5). L-Trp also serves as a precursor for the synthesis of the neurotransmitter serotonin and the hormone melatonin. IDO exhibits a broader substrate specificity than TDO, because the former can degrade indoleamines, including L-Trp, D-Trp, serotonin, melatonin, and tryptamine (6). In addition to its role as a L-Trp-catabolizing enzyme, IDO is involved in the immunoregulating system [review by Mellor and M...

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Heme oxygenase (HO) catalyzes the O(2)- and NADPH-cytochrome P450 reductase-dependent conversion of heme to biliverdin, Fe, and CO through a process in which the heme participates both as a prosthetic group and as a substrate. In the present study, we have generated a detailed reaction cycle for the first monooxygenation step of HO catalysis, conversion of the heme to alpha-meso-hydroxyheme. We employed EPR (using both (16)O(2) and (17)O(2)) and (1)H, (14)N ENDOR spectroscopies to characterize the intermediates generated by 77 K radiolytic cryoreduction and subsequent annealing of wild-type oxy-HO and D140A, F mutants. One-electron cryoreduction of oxy-HO yields a hydroperoxoferri-HO with g-tensor, g = [2.37, 2.187, 1.924]. Annealing of this species to 200 K is accompanied by spectroscopic changes that include the appearance of a new (1)H ENDOR signal, reflecting rearrangements in the active site. Kinetic measurements at 214 K reveal that the annealed hydroperoxoferri-HO species, denoted R, generates the ferri-alpha-meso-hydroxyheme product in a first-order reaction. Disruption of the H-bonding network within the distal pocket of HO by the alanine and phenylalanine mutations of residue D140 prevents product formation. The hydroperoxoferri-HO (D140A) instead undergoes heterolytic cleavage of the O-O bond, ultimately yielding an EPR-silent compound II-like species that does not form product. These results, which agree with earlier suggestions, establish that hydroperoxoferri-HO is indeed the reactive species, directly forming the alpha-meso-hydroxyheme product by attack of the distal OH of the hydroperoxo moiety at the heme alpha-carbon.