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Probing the ternary complexes of indoleamine and tryptophan 2,3-dioxygenases by cryoreduction EPR and ENDOR spectroscopy.

Davydov RM, Chauhan N, Thackray SJ, Anderson JL, Papadopoulou ND, Mowat CG, Chapman SK, Raven EL, Hoffman BM - J. Am. Chem. Soc. (2010)

Bottom Line: The results reveal the presence of multiple conformations of the binary ferrous-oxy species of the IDOs.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 O(2) into the C(2)-C(3) double bond of the substrate.This substrate interaction further helps control the reactivity of the heme-bound dioxygen by "shielding" it from water.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Northwestern University, Evanston, Illinois 60208, USA.

ABSTRACT
We have applied cryoreduction/EPR/ENDOR techniques to characterize the active-site structure of the ferrous-oxy complexes of human (hIDO) and Shewanella oneidensis (sIDO) indoleamine 2,3-dioxygenases, Xanthomonas campestris (XcTDO) tryptophan 2,3-dioxygenase, and the H55S variant of XcTDO 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-O(2)-Trp] dioxygenase complexes generates a ferric peroxy heme species with very similar EPR and (1)H 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 O(2), 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 O(2) into the C(2)-C(3) double bond of the substrate. This substrate interaction further helps control the reactivity of the heme-bound dioxygen by "shielding" it from water.

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Model of the [XcTDOII-O2-Trp] ternary complex, based on PDB entry 2NW8. Bond distances are shown in italics, l-Trp is shown in magenta, and dioxygen is shown in red. (A) Side view, with heme shown in gray. (B) Overhead view, with heme shown in white. The distance between the N of the Trp ammonium group and the distal O of O2 is ∼2.9 Å.
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fig5: Model of the [XcTDOII-O2-Trp] ternary complex, based on PDB entry 2NW8. Bond distances are shown in italics, l-Trp is shown in magenta, and dioxygen is shown in red. (A) Side view, with heme shown in gray. (B) Overhead view, with heme shown in white. The distance between the N of the Trp ammonium group and the distal O of O2 is ∼2.9 Å.

Mentions: Examination of the structure for substrate-bound ferrous and ferric XcTDO(12) as well as a model created for the ternary complex11,12 instead leads us to suggest that the hydrogen bond to the bound O2 detected by cryoreduction/1H ENDOR for all the ternary complexes with l-Trp and Me-Trp likely can be assigned to a proton of the ammonium group of the substrate, which is situated within hydrogen-bonding distance (Figure 5). This interaction would be analogous to the hydrogen bond between the distal histidine and the O2 ligand in the globins.(28) It would be consistent with our observations12,29 that the substrate analogue indole propionic acid, in which the amine group is missing, does not form product. In principle, it would be possible to test our proposal with indole propionic acid, but unfortunately this was unsuccessful because the ferrous-oxy complex for this substrate analogue is unstable through autoxidation and could not be trapped for the EPR/ENDOR experiments. This proposal would also provide an explanation for results suggesting superoxide character of the heme-bound O2 in the ternary complex,16,34 because charge polarization would be favored by hydrogen-bonding and electrostatic interactions between the positive-charged ammonium group of the substrate and the distal oxygen of bound O2. It also correlates with the reported appearance of a positive electrostatic potential surrounding the heme-bound CO in hIDO when l-Trp is present and the specific effect of the −CH(NH3)+COO− moiety of l-Trp or its analogues on the stretching frequencies of bound CO in rat liver TDO.13,15,35


Probing the ternary complexes of indoleamine and tryptophan 2,3-dioxygenases by cryoreduction EPR and ENDOR spectroscopy.

Davydov RM, Chauhan N, Thackray SJ, Anderson JL, Papadopoulou ND, Mowat CG, Chapman SK, Raven EL, Hoffman BM - J. Am. Chem. Soc. (2010)

Model of the [XcTDOII-O2-Trp] ternary complex, based on PDB entry 2NW8. Bond distances are shown in italics, l-Trp is shown in magenta, and dioxygen is shown in red. (A) Side view, with heme shown in gray. (B) Overhead view, with heme shown in white. The distance between the N of the Trp ammonium group and the distal O of O2 is ∼2.9 Å.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2903012&req=5

fig5: Model of the [XcTDOII-O2-Trp] ternary complex, based on PDB entry 2NW8. Bond distances are shown in italics, l-Trp is shown in magenta, and dioxygen is shown in red. (A) Side view, with heme shown in gray. (B) Overhead view, with heme shown in white. The distance between the N of the Trp ammonium group and the distal O of O2 is ∼2.9 Å.
Mentions: Examination of the structure for substrate-bound ferrous and ferric XcTDO(12) as well as a model created for the ternary complex11,12 instead leads us to suggest that the hydrogen bond to the bound O2 detected by cryoreduction/1H ENDOR for all the ternary complexes with l-Trp and Me-Trp likely can be assigned to a proton of the ammonium group of the substrate, which is situated within hydrogen-bonding distance (Figure 5). This interaction would be analogous to the hydrogen bond between the distal histidine and the O2 ligand in the globins.(28) It would be consistent with our observations12,29 that the substrate analogue indole propionic acid, in which the amine group is missing, does not form product. In principle, it would be possible to test our proposal with indole propionic acid, but unfortunately this was unsuccessful because the ferrous-oxy complex for this substrate analogue is unstable through autoxidation and could not be trapped for the EPR/ENDOR experiments. This proposal would also provide an explanation for results suggesting superoxide character of the heme-bound O2 in the ternary complex,16,34 because charge polarization would be favored by hydrogen-bonding and electrostatic interactions between the positive-charged ammonium group of the substrate and the distal oxygen of bound O2. It also correlates with the reported appearance of a positive electrostatic potential surrounding the heme-bound CO in hIDO when l-Trp is present and the specific effect of the −CH(NH3)+COO− moiety of l-Trp or its analogues on the stretching frequencies of bound CO in rat liver TDO.13,15,35

Bottom Line: The results reveal the presence of multiple conformations of the binary ferrous-oxy species of the IDOs.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 O(2) into the C(2)-C(3) double bond of the substrate.This substrate interaction further helps control the reactivity of the heme-bound dioxygen by "shielding" it from water.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Northwestern University, Evanston, Illinois 60208, USA.

ABSTRACT
We have applied cryoreduction/EPR/ENDOR techniques to characterize the active-site structure of the ferrous-oxy complexes of human (hIDO) and Shewanella oneidensis (sIDO) indoleamine 2,3-dioxygenases, Xanthomonas campestris (XcTDO) tryptophan 2,3-dioxygenase, and the H55S variant of XcTDO 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-O(2)-Trp] dioxygenase complexes generates a ferric peroxy heme species with very similar EPR and (1)H 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 O(2), 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 O(2) into the C(2)-C(3) double bond of the substrate. This substrate interaction further helps control the reactivity of the heme-bound dioxygen by "shielding" it from water.

Show MeSH
Related in: MedlinePlus