Probing the Ternary Complexes of Indoleamine and Tryptophan 2,3-Dioxygenases by Cryoreduction EPR and ENDOR Spectroscopy

• Published in: Journal of the American Chemical Society Vol. 132; no. 15; pp. 5494 - 5500
• Main Authors: Davydov, Roman M, Chauhan, Nishma, Thackray, Sarah J, Anderson, J. L. Ross, Papadopoulou, Nektaria D, Mowat, Christopher G, Chapman, Stephen K, Raven, Emma L, Hoffman, Brian M
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 21.04.2010
• Subjects: Bonding; Catalytic Domain; Electron Spin Resonance Spectroscopy - methods; Ferrous Compounds - metabolism; Humans; Hydrogen Bonding; Hydrogen bonds; Indoleamine-Pyrrole 2,3,-Dioxygenase - chemistry; Indoleamine-Pyrrole 2,3,-Dioxygenase - metabolism; Indoles; Ligands; Oxygen - chemistry; Protonation; Shewanella oneidensis; Spectra; Tryptophan; Tryptophan - metabolism; Tryptophan Oxygenase - chemistry; Tryptophan Oxygenase - metabolism; Xanthomonas campestris; Xanthomonas campestris - enzymology
• ISSN: 0002-7863; 1520-5126; 1520-5126
• DOI: 10.1021/ja100518z

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.