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

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    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
    Issue number15
    Publication statusPublished - 21 Apr 2010


    • P450CAM
    • HEME
    • FERRYL


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