Extensive studies of the heme coordination structure of indoleamine 2,3-dioxygenase and of tryptophan binding with magnetic and natural circular dichroism and electron paramagnetic resonance spectroscopy.
In order to probe the active site of the heme protein indoleamine 2,3-dioxygenase, magnetic and natural circular dichroism (MCD and CD) and electron paramagnetic resonance (EPR) studies of the substrate (L-tryptophan)-free and substrate-bound enzyme with and without various exogenous ligands have been carried out. The MCD spectra of the ferric and ferrous derivatives are similar to those of the analogous myoglobin and horseradish peroxidase species. This provides strong support for histidine imidazole as the fifth ligand to the heme iron of indoleamine 2,3-dioxygenase. The substrate-free native ferric enzyme exhibits predominantly high-spin EPR signals (g perpendicular = 6, g parallel = 2) along with weak low-spin signals (g perpendicular = 2.86, 2.28, 1.60); similar EPR, spin-state and MCD features are found for the benzimidazole adduct of ferric myoglobin. This suggests that the substrate-free ferric enzyme has a sterically hindered histidine imidazole nitrogen donor sixth ligand. Upon substrate binding, noticeable MCD and EPR spectral changes are detected that are indicative of an increased low spin content (from 30 to over 70% at ambient temperature). Concomitantly, new low spin EPR signals (g = 2.53, 2.18, 1.86) and MCD features characteristic of hydroxide complexes of histidine-ligated heme proteins appear. For almost all of the other ferric and ferrous derivatives, only small substrate effects are observed with MCD spectroscopy, while substantial substrate effects are seen with CD spectroscopy. Thus, changes in the heme coordination structure of the ferric enzyme and in the protein conformation at the active site of the ferric and ferrous enzyme are induced by substrate binding. The observed substrate effects on the ferric enzyme may correlate with the previously observed kinetic substrate inhibition of indoleamine 2,3-dioxygenase activity, while such effects on the ferrous enzyme suggest the possibility that the substrate is activated during turnover.
D000818:Animals; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D005290:Ferric Compounds / Q000378:metabolism; D005296:Ferrous Compounds / Q000378:metabolism; D006418:Heme / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007421:Intestine, Small / Q000201:enzymology; D010105:Oxygenases / Q000378:metabolism*; D011817:Rabbits; D014364:Tryptophan / Q000378:metabolism*; D014366:Tryptophan Oxygenase
D005290:Ferric Compounds; D005296:Ferrous Compounds; D006418:Heme; D014364:Tryptophan; D010105:Oxygenases; D014366:Tryptophan Oxygenase