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

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• 作者：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
• 刊名：Journal of the American Chemical Society
• 出版年：2010
• 出版时间：April 21, 2010
• 年：2010
• 卷：132
• 期：15
• 页码：5494-5500
• 全文大小：241K
• 年卷期：v.132,no.15(April 21, 2010)
• ISSN：1520-5126

文摘

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.