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 L. Raven, Brian M. Hoffman

Research output: Contribution to journalArticlepeer-review

37 Scopus citations

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.

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

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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