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

Roman M Davydov, Nishma Chauhan, Sarah J Thackray, J L Ross Anderson, Nektaria D Papadopoulou, Christopher G Mowat, Stephen K Chapman, Emma Lloyd Raven, Brian M Hoffman

    Research output: Contribution to journalArticle

    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-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 C 2-C3 double bond of the substrate. This substrate interaction further helps control the reactivity of the heme-bound dioxygen by shielding it from water. © 2010 American Chemical Society.

    Original languageEnglish
    Pages (from-to)5494-5500
    Number of pages7
    JournalJournal of the American Chemical Society
    Volume132
    Issue number15
    DOIs
    Publication statusPublished - 21 Apr 2010

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    Tryptophan Oxygenase
    Indoleamine-Pyrrole 2,3,-Dioxygenase
    Electron Spin Resonance Spectroscopy
    Spectrum Analysis
    Oxygen
    Ligands
    Heme
    Hydrogen
    Shewanella
    Xanthomonas campestris
    Dioxygenases
    Water
    Hydrogen Bonding
    Ammonium Compounds
    Protons
    Catalytic Domain
    Nitrogen
    Enzymes
    indole

    Keywords

    • BINDING
    • P450CAM
    • PROTEIN
    • HEME
    • CATALYTIC MECHANISM
    • NUCLEAR DOUBLE-RESONANCE
    • AMINO-ACID-SEQUENCE
    • CRYSTAL-STRUCTURE
    • FERRYL
    • DIOXYGENASE-LIKE-MYOGLOBIN

    Cite this

    Davydov, R. M., Chauhan, N., Thackray, S. J., Anderson, J. L. R., Papadopoulou, N. D., Mowat, C. G., ... Hoffman, B. M. (2010). Probing the ternary complexes of indoleamine and tryptophan 2,3-dioxygenases by cryoreduction EPR and ENDOR spectroscopy. Journal of the American Chemical Society, 132(15), 5494-5500. https://doi.org/10.1021/ja100518z
    Davydov, Roman M ; Chauhan, Nishma ; Thackray, Sarah J ; Anderson, J L Ross ; Papadopoulou, Nektaria D ; Mowat, Christopher G ; Chapman, Stephen K ; Raven, Emma Lloyd ; Hoffman, Brian M. / Probing the ternary complexes of indoleamine and tryptophan 2,3-dioxygenases by cryoreduction EPR and ENDOR spectroscopy. In: Journal of the American Chemical Society. 2010 ; Vol. 132, No. 15. pp. 5494-5500.
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    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-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 C 2-C3 double bond of the substrate. This substrate interaction further helps control the reactivity of the heme-bound dioxygen by shielding it from water. {\circledC} 2010 American Chemical Society.",
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    author = "Davydov, {Roman M} and Nishma Chauhan and Thackray, {Sarah J} and Anderson, {J L Ross} and Papadopoulou, {Nektaria D} and Mowat, {Christopher G} and Chapman, {Stephen K} and Raven, {Emma Lloyd} and Hoffman, {Brian M}",
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    Davydov, RM, Chauhan, N, Thackray, SJ, Anderson, JLR, Papadopoulou, ND, Mowat, CG, Chapman, SK, Raven, EL & Hoffman, BM 2010, 'Probing the ternary complexes of indoleamine and tryptophan 2,3-dioxygenases by cryoreduction EPR and ENDOR spectroscopy', Journal of the American Chemical Society, vol. 132, no. 15, pp. 5494-5500. https://doi.org/10.1021/ja100518z

    Probing the ternary complexes of indoleamine and tryptophan 2,3-dioxygenases by cryoreduction EPR and ENDOR spectroscopy. / Davydov, Roman M; Chauhan, Nishma; Thackray, Sarah J; Anderson, J L Ross; Papadopoulou, Nektaria D; Mowat, Christopher G; Chapman, Stephen K; Raven, Emma Lloyd; Hoffman, Brian M.

    In: Journal of the American Chemical Society, Vol. 132, No. 15, 21.04.2010, p. 5494-5500.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Probing the ternary complexes of indoleamine and tryptophan 2,3-dioxygenases by cryoreduction EPR and ENDOR spectroscopy

    AU - Davydov, Roman M

    AU - Chauhan, Nishma

    AU - Thackray, Sarah J

    AU - Anderson, J L Ross

    AU - Papadopoulou, Nektaria D

    AU - Mowat, Christopher G

    AU - Chapman, Stephen K

    AU - Raven, Emma Lloyd

    AU - Hoffman, Brian M

    PY - 2010/4/21

    Y1 - 2010/4/21

    N2 - 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-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 C 2-C3 double bond of the substrate. This substrate interaction further helps control the reactivity of the heme-bound dioxygen by shielding it from water. © 2010 American Chemical Society.

    AB - 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-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 C 2-C3 double bond of the substrate. This substrate interaction further helps control the reactivity of the heme-bound dioxygen by shielding it from water. © 2010 American Chemical Society.

    KW - BINDING

    KW - P450CAM

    KW - PROTEIN

    KW - HEME

    KW - CATALYTIC MECHANISM

    KW - NUCLEAR DOUBLE-RESONANCE

    KW - AMINO-ACID-SEQUENCE

    KW - CRYSTAL-STRUCTURE

    KW - FERRYL

    KW - DIOXYGENASE-LIKE-MYOGLOBIN

    UR - http://www.scopus.com/inward/record.url?scp=77951041547&partnerID=8YFLogxK

    U2 - 10.1021/ja100518z

    DO - 10.1021/ja100518z

    M3 - Article

    C2 - 20353179

    VL - 132

    SP - 5494

    EP - 5500

    JO - Journal of the American Chemical Society

    JF - Journal of the American Chemical Society

    SN - 0002-7863

    IS - 15

    ER -