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

R.M Davydov, N Chauhan, S.J Thackray, J.L.R Anderson, N.D Papadopoulou, N.D Mowat, S.K Chapman, E.L Raven, B.M Hoffman

Research output: Contribution to journalArticle (Academic Journal)

37 Citations (Scopus)

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 C2-C3 double bond of the substrate. This substrate interaction further helps control the reactivity of the heme-bound dioxygen by “shielding” it from water.
Translated title of the contributionProbing the ternary complexes of indoleamine and tryptophan 2,3-dioxygenases by cryoreduction EPR and ENDOR spectroscopy
Original languageEnglish
Pages (from-to)5494 - 5500
Number of pages7
JournalJournal of the American Chemical Society
Volume132
Issue number15
DOIs
Publication statusPublished - Apr 2010

Fingerprint Dive into the research topics of 'Probing the ternary complexes of indoleamine and tryptophan 2,3-dioxygenases by cryoreduction EPR and ENDOR spectroscopy'. Together they form a unique fingerprint.

Cite this