Idoleamine 2,3-dioxygenase (IDO) is one of the few known heme-containing dioxygenases. It catalyzes the oxidative cleavage of the indole ring of tryptophan and its analogs (indoleamines) by incorporating O2 into organic substrates. Although tryptophan 2,3-dioxygenase (TPO)[liver and bacterial (Pseudomonas acidovorans)] catalyzes the same reaction, IDO is distinct from TPO: IDO is ubiquitously distribut in animal tissues except the liver. Moreover, a broad substrate specificity, the requirement of reductant co-factors, and most noticeably, a high reactivity toward superoxide anion radical (O2-.), a toxic oxygen species know to be responsible for direct and indirect tissue damage, are unique properties of IDO. On the basis of the last property and the enzyme's ability to metabolize a neurotransmitter, serotonin, the following two possible physiological roles of IDO have been suggested: (a) an O2-. scavenger and (b) a regulator of serotonin. Previous studies have yet to establish the validity of the above speculations. The IDO.substrate.O2 ternary complex is highly autoxidizable and is easily converted to the ferric (inactive) form. Thus, either an ascorbic acid-methylene blue or O2-.-methylene blue system is required as reductant co-factors in vitro. The natural co-factors have not been identified. In order to clarify the current ambiguities regarding the physiological role of IDO through studies of its active site structure and reaction mechanism, the present research project, using purified rabbit intestinal IDO, will set the following specific aims. (1) Equilibrium and kinetic interactions of IDO with organic substrates (indoleamines), O2, and inhibitors will be studied with various applicable spectroscopic techniques (UV- visible, EPR, CD, NMR and stopped flow). (2) Using an enzymatic O2-.- generating system and superoxide dismutase, an O2-. scavenger, the participation of the co-factors in the enzyme activation will be quantitatively examined. (3) The direct reaction, ferric IDO + O2-.--greater than IDO.O2, will be studied in detail by pulse radiolysis. (4) Using a relatively new technique, optically transparent thin-layer electrolysis (OTTLE), the reduction potential (Eomicron') of IDO will be determined. (5) The role of the heme iron prosthetic group of IDO in the incorporation of O2 into substrate, in comparison with that of metals (Co and Mn) used for dioxygenase model complexes studied by others, will be investigated by heme and metal substitution techniques using chemically modified porphyrins with Fe, Co, Ru ets.
