Probing the ternary complexes of indoleamine and tryptophan 2,3-dioxygenases by cryoreduction EPR and ENDOR spectroscopy.
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-O(2)-Trp] dioxygenase complexes generates a ferric peroxy heme species with very similar EPR and (1)H 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 O(2), 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 O(2) into the C(2)-C(3) double bond of the substrate. This substrate interaction further helps control the reactivity of the heme-bound dioxygen by "shielding" it from water.
Davydov
Roman M
RM
Department of Chemistry, Northwestern University, Evanston, Illinois 60208, USA.
Chauhan
Nishma
N
Thackray
Sarah J
SJ
Anderson
J L Ross
JL
Papadopoulou
Nektaria D
ND
Mowat
Christopher G
CG
Chapman
Stephen K
SK
Raven
Emma L
EL
Hoffman
Brian M
BM
D020134:Catalytic Domain; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D005296:Ferrous Compounds / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase / Q000737:chemistry* / Q000378:metabolism; D010100:Oxygen / Q000737:chemistry; D014364:Tryptophan / Q000378:metabolism; D014366:Tryptophan Oxygenase / Q000737:chemistry* / Q000378:metabolism; D016959:Xanthomonas campestris / Q000201:enzymology
D005296:Ferrous Compounds; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D014364:Tryptophan; D014366:Tryptophan Oxygenase; D010100:Oxygen