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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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1H ENDOR spectra for cryoreduced oxy hIDO (species B) and ternary complexes of oxy IDO, XcTDO, and oxy H55S XcTDO with Trp, Me-Trp, and 5F-Trp. Instrument conditions: T = 2 K; microwave frequency, 34.95 GHz; modulation amplitude, 2 G; rf power, 5 W; rf sweep rate, 0.5 MHz/s; rf broadening, 60 kHz; 20 scans.
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fig3: 1H ENDOR spectra for cryoreduced oxy hIDO (species B) and ternary complexes of oxy IDO, XcTDO, and oxy H55S XcTDO with Trp, Me-Trp, and 5F-Trp. Instrument conditions: T = 2 K; microwave frequency, 34.95 GHz; modulation amplitude, 2 G; rf power, 5 W; rf sweep rate, 0.5 MHz/s; rf broadening, 60 kHz; 20 scans.

Mentions: In contrast to the ferrous-oxy case above, the EPR spectrum of cryoreduced [hIDOII-O2-Trp] displays just a single new EPR signal, with g = [2.270, 2.170, 1.946] (Figure 1, Table 1), a g-tensor characteristic of a ferric peroxy heme intermediate.19,21,26 This assignment is corroborated by 1H ENDOR data. This intermediate displays a strongly coupled exchangeable proton ENDOR signal, Amax ≈ 15 MHz (Figures 3, S6), the value commonly observed for ferric peroxy heme species.(19) This result for the substrate-bound dioxygenases contrasts with those for heme monooxygenases, whose ternary oxy complexes directly form the ferric hydroperoxy state, in which the proton of the hydroperoxy ligand has a smaller hyperfine coupling, Amax ≤ 12 MHz.18,21


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)

1H ENDOR spectra for cryoreduced oxy hIDO (species B) and ternary complexes of oxy IDO, XcTDO, and oxy H55S XcTDO with Trp, Me-Trp, and 5F-Trp. Instrument conditions: T = 2 K; microwave frequency, 34.95 GHz; modulation amplitude, 2 G; rf power, 5 W; rf sweep rate, 0.5 MHz/s; rf broadening, 60 kHz; 20 scans.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: 1H ENDOR spectra for cryoreduced oxy hIDO (species B) and ternary complexes of oxy IDO, XcTDO, and oxy H55S XcTDO with Trp, Me-Trp, and 5F-Trp. Instrument conditions: T = 2 K; microwave frequency, 34.95 GHz; modulation amplitude, 2 G; rf power, 5 W; rf sweep rate, 0.5 MHz/s; rf broadening, 60 kHz; 20 scans.
Mentions: In contrast to the ferrous-oxy case above, the EPR spectrum of cryoreduced [hIDOII-O2-Trp] displays just a single new EPR signal, with g = [2.270, 2.170, 1.946] (Figure 1, Table 1), a g-tensor characteristic of a ferric peroxy heme intermediate.19,21,26 This assignment is corroborated by 1H ENDOR data. This intermediate displays a strongly coupled exchangeable proton ENDOR signal, Amax ≈ 15 MHz (Figures 3, S6), the value commonly observed for ferric peroxy heme species.(19) This result for the substrate-bound dioxygenases contrasts with those for heme monooxygenases, whose ternary oxy complexes directly form the ferric hydroperoxy state, in which the proton of the hydroperoxy ligand has a smaller hyperfine coupling, Amax ≤ 12 MHz.18,21

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