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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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X-band EPR spectra of cryoreduced binary ferrous-oxy hIDO and sIDO complexes and cryoreduced ternary complexes of ferrous-oxy hIDO, XcTDO, and H55S XcTDO with Trp, Me-Trp, and 5F-Trp. Instrument conditions: T = 25 K; microwave power, 2 mW; modulation amplitude, 5 G; microwave frequency, 9.365 GHz.
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fig1: X-band EPR spectra of cryoreduced binary ferrous-oxy hIDO and sIDO complexes and cryoreduced ternary complexes of ferrous-oxy hIDO, XcTDO, and H55S XcTDO with Trp, Me-Trp, and 5F-Trp. Instrument conditions: T = 25 K; microwave power, 2 mW; modulation amplitude, 5 G; microwave frequency, 9.365 GHz.

Mentions: The EPR spectrum of ferrous-oxy hIDO [hIDOII-O2] which was radiolytically reduced at 77 K is presented in Figure 1. The spectrum is a superposition of multiple rhombic S = 1/2 EPR signals with a range of gmax-values, 2.35 ≤ gmax ≤ 2.20. Its shape is independent of the degree of cryoreduction, indicating that it is determined by the presence of multiple conformers of the parent diamagnetic ferrous-oxy complex, and not by the relative probabilities of reduction of the conformers of the oxy heme parents. Two signals stand out as perhaps more intense and thus representing more highly populated parent conformers; they are characterized by g = [2.32, 2.17, 1.95] (signal A) and g = [2.348, 2.26, 1.922] (signal B) (Table 1), values that are typical of a ferric hydroperoxy heme species (Fe(III)-OOH−).19,21,26 A minority of the conformers have signals with gmax ≤ 2.26, characteristic of cryogenerated ferric peroxy heme (Fe(III)-OO2−) intermediates. The assignment of species A and B as ferric hydroperoxy species was corroborated by 1H ENDOR spectroscopy. The trapped A and B species show exchangeable 1H ENDOR spectra with Amax = 12 and 10 MHz, respectively (Figure S1, Supporting Information), values that are characteristic of the hydroperoxy ligand.(18)


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)

X-band EPR spectra of cryoreduced binary ferrous-oxy hIDO and sIDO complexes and cryoreduced ternary complexes of ferrous-oxy hIDO, XcTDO, and H55S XcTDO with Trp, Me-Trp, and 5F-Trp. Instrument conditions: T = 25 K; microwave power, 2 mW; modulation amplitude, 5 G; microwave frequency, 9.365 GHz.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: X-band EPR spectra of cryoreduced binary ferrous-oxy hIDO and sIDO complexes and cryoreduced ternary complexes of ferrous-oxy hIDO, XcTDO, and H55S XcTDO with Trp, Me-Trp, and 5F-Trp. Instrument conditions: T = 25 K; microwave power, 2 mW; modulation amplitude, 5 G; microwave frequency, 9.365 GHz.
Mentions: The EPR spectrum of ferrous-oxy hIDO [hIDOII-O2] which was radiolytically reduced at 77 K is presented in Figure 1. The spectrum is a superposition of multiple rhombic S = 1/2 EPR signals with a range of gmax-values, 2.35 ≤ gmax ≤ 2.20. Its shape is independent of the degree of cryoreduction, indicating that it is determined by the presence of multiple conformers of the parent diamagnetic ferrous-oxy complex, and not by the relative probabilities of reduction of the conformers of the oxy heme parents. Two signals stand out as perhaps more intense and thus representing more highly populated parent conformers; they are characterized by g = [2.32, 2.17, 1.95] (signal A) and g = [2.348, 2.26, 1.922] (signal B) (Table 1), values that are typical of a ferric hydroperoxy heme species (Fe(III)-OOH−).19,21,26 A minority of the conformers have signals with gmax ≤ 2.26, characteristic of cryogenerated ferric peroxy heme (Fe(III)-OO2−) intermediates. The assignment of species A and B as ferric hydroperoxy species was corroborated by 1H ENDOR spectroscopy. The trapped A and B species show exchangeable 1H ENDOR spectra with Amax = 12 and 10 MHz, respectively (Figure S1, Supporting Information), values that are characteristic of the hydroperoxy ligand.(18)

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