Human indoleamine 2,3-Dioxygenase

We have developed an efficient bacterial expression system for production of human IDO (hIDO) that utilizes a His-tag expression vector and have established reliable protocols for purification of the recombinant protein. We have used this expression system to examine the redox, spectroscopic and substrate-binding properties of the enzyme. The Fe3+/Fe2+ reduction potential was found to be -30 +/- 4 mV; in the presence of L-Trp, this value increases to + 16 +/- 3 mV. Electronic, EPR and MCD spectroscopies indicate that ferric rhIDO (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. 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.;The role of two key histidine residues, H346A and H303A, was also examined in Chapter 4. Parallel spectroscopic, electrochemical and ligand binding analyses were consistent either with H303 as the sixth ligand or with H303 linked to a conformational change that affects the formation of the low-spin heme species. The first scenario is ruled out in light of the recently published crystal structure of rhIDO. Further analyses of the H303A variant indicated that this residue is not required for the formation of the low-spin, hydroxide-bound heme in the presence of the substrate. The Fe3+/Fe2+ reduction potential of H303A variant is "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

Probing the Ternary Complexes of Indoleamine and Tryptophan 2,3-Dioxygenases by Cryoreduction EPR and ENDOR Spectroscopy

We have applied cryoreduction/EPR/ENDOR techniques to characterize the active-site structure of the ferrous-oxy complexes of human (hIDO) and <i>Shewanella oneidensis</i> (sIDO) indoleamine 2,3-dioxygenases, <i>Xanthomonas campestris</i> (<i>Xc</i>TDO) tryptophan 2,3-dioxygenase, and the H55S variant of <i>Xc</i>TDO in the absence and in the presence of the substrate l-Trp and a substrate analogue, l-Me-Trp. The results reveal the presence of multiple conformations of the binary ferrous-oxy species of the IDOs. In more populated conformers, most likely a water molecule is within hydrogen-bonding distance of the bound ligand, which favors protonation of a cryogenerated ferric peroxy species at 77 K. In contrast to the binary complexes, cryoreduction of all of the studied ternary [enzyme-O₂-Trp] dioxygenase complexes generates a ferric peroxy heme species with very similar EPR and ¹H ENDOR spectra in which protonation of the basic peroxy ligand does not occur at 77 K. Parallel studies with l-Me-Trp, in which the proton of the indole nitrogen is replaced with a methyl group, eliminate the possibility that the indole NH group of the substrate acts as a hydrogen bond donor to the bound O₂, and we suggest instead that the ammonium group of the substrate hydrogen-bonds to the dioxygen ligand. The present data show that substrate binding, primarily through this H-bond, causes the bound dioxygen to adopt a new conformation, which presumably is oriented for insertion of O₂ into the C₂−C₃ double bond of the substrate. This substrate interaction further helps control the reactivity of the heme-bound dioxygen by “shielding” it from water