l-Tryptophan 2,3-dioxygenase (TDO) is a protoheme-containing enzyme that catalyzes the production of N-formylkynurenine by inserting O2 into the pyrrole ring of l-tryptophan. Although a ferrous–oxy form (Fe2+–O2) has been established to be an obligate intermediate in the reaction, details of the ring opening reaction remain elusive. In this study, the O2 insertion reaction catalyzed by Pseudomonas TDO (PaTDO) was examined using a heme-modification approach, which allowed us to draw a quantitative correlation between the inductive electronic effects of the heme substituents and the substituent-induced changes in the functional behaviors of the ferrous–oxy form. We succeeded in preparing reconstituted PaTDO with synthetic hemes, which were different with respect to the inductive electron-withdrawing nature of the heme substituents at positions 2 and 4. An increase in the electron-withdrawing power of the heme substituents elevated the redox potential of reconstituted PaTDO, showing that the stronger the electron-withdrawing ability of the heme substituents, the lower the electron density on the heme iron. The decrease in the electron density of the heme iron resulted in a higher frequency shift of the C–O stretch of the heme-bound CO and enhanced the dissociation of O2 from the ferrous–oxy intermediate. This result was interpreted as being due to weaker π back-donation from the heme iron to the bound CO or O2. More importantly, the reaction rates of the ferrous–oxy intermediate to oxidize l-Trp were increased with the electron-withdrawing ability of the heme substituents, implying that the more electron-deficient ferrous–oxy heme is favored for the PaTDO-catalyzed oxygenation. On the basis of these results, we propose that the initial step of the dioxygen activation by PaTDO is a direct electrophilic addition of the heme-bound O2 to the indole ring of l-Trp.

Nitric oxide (NO) elicits a wide variety of physiological responses by binding to the heme in soluble guanylate cyclase (sGC) to stimulate cGMP production. Although nucleotides, such as ATP or GTP analogues, have been reported to regulate the signaling of NO binding from the heme site to the catalytic site, the other regulatory functions of nucleotides remain unexamined. Among the nucleotides tested, we found that 2′-d-3′-GMP acted as a potent noncompetitive inhibitor with respect to Mn-GTP, when the ferrous enzyme combined with NO, CO, or allosteric activator BAY 41-2272. 2′-d-3′-GMP also displayed nearly identical patterns of inhibition for the ferric enzyme, in which the binding of N3– or BAY 41-2272 significantly increased the inhibitory effects of the nucleotide. Equilibrium dialysis measurements using the CO-ligated enzyme in the presence of allosteric activators demonstrated that 2′-d-3′-GMP exclusively binds to the catalytic site of sGC. Furthermore, the affinity of 2′-d-3′-GMP for the enzyme was found to increase upon addition of foscarnet, an analogue of PPi. These findings together with other kinetic results imply that 2′-d-3′-GMP acts as a P-site inhibitor probably by forming a dead-end complex, sGC–2′-d-3′-GMP–PPi, in the catalytic reaction. The formation of the complex of the enzyme with 2′-d-3′-GMP does not seem to be associated with changes in the Fe–proximal His bond strength, because the CO coordination state or the redox potentials of the enzyme-heme complex are virtually unaffected.

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 (O2). 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 α-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 substrate-binding affinity, and an electron density map reveals that 2-(N-cyclohexylamino)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.

We determined the structure of the complex of the sensory histidine kinase (HK) and its cognate response regulator (RR) in the two-component signal transduction system of Thermotoga maritima. This was accomplished by fitting the high-resolution structures of the isolated HK domains and the RR onto the electron density map (3.8 Å resolution) of the HK/RR complex crystal. Based on the structural information, we evaluated the roles of both interdomain and intermolecular interactions in the signal transduction of the cytosolic PAS-linked HK and RR system, in particular the O2-sensor FixL/FixJ system. The PAS-sensor domain of HK interacts with the catalytic domain of the same polypeptide chain by creating an interdomain β sheet. The interaction site between HK and RR, which was confirmed by NMR, is suitable for the intermolecular transfer reaction of the phosphoryl group, indicating that the observed interaction is important for the phosphatase activity of HK that dephosphorylates phospho-RR.