TY - JOUR
T1 - Probing the ternary complexes of indoleamine and tryptophan 2,3-dioxygenases by cryoreduction epr and endor spectroscopy
AU - Davydov, Roman M.
AU - Chauhan, Nishma
AU - Thackray, Sarah J.
AU - Anderson, J. L.Ross
AU - Papadopoulou, Nektaria D.
AU - Mowat, Christopher G.
AU - Chapman, Stephen K.
AU - Raven, Emma L.
AU - Hoffman, Brian M.
PY - 2010/4/21
Y1 - 2010/4/21
N2 - 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.
AB - 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.
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U2 - 10.1021/ja100518z
DO - 10.1021/ja100518z
M3 - Article
C2 - 20353179
AN - SCOPUS:77951041547
VL - 132
SP - 5494
EP - 5500
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
SN - 0002-7863
IS - 15
ER -