The dioxygen adduct of the heme protein indoleamine 2,3-dioxygenase has been generated at -30 degrees C in mixed solvents, and spectroscopic and equilibrium studies of its L-tryptophan (substrate) binding properties have been carried out for the first time. Comparative studies have also been performed with the NO and CO adducts of the ferrous enzyme. Under the conditions employed (-30 degrees C), both autoxidation and turnover (L-tryptophan + O2----formylkynurenine) of the ternary complex are effectively suppressed. Structural identification of the ternary complex is based on the 1:1 molar stoichiometry for the substrate-oxygenated enzyme adduct formation (Kd approximately 10(-4) M), the time-dependent linear product formation (turnover) at -20 degrees C, and the quantitative conversion of the complex to the ferrous CO derivative by bubbling with CO. Binding of L-tryptophan to the oxygenated enzyme leads to decreases in the intensities of its major absorption bands (lambda max 415, 541, 576 nm) and to a blue shift of its Soret peak. Interestingly, among the ferrous enzyme derivatives examined, only the substrate-bound oxygenated enzyme exhibits solvent-dependent Soret absorption peak positions, e.g., lambda max 411.5 and 413.5 nm in 65% (v/v) aqueous glycerol and ethylene glycol, respectively. In addition, indole binds to the oxygenated enzyme, causing a red shift of its Soret peak in these solvents only in the presence of substrate (411.5----414 nm and 413.5----414.5 nm, respectively), while similar effects of indole are independent of tryptophan for the other ferrous enzyme derivatives.(ABSTRACT TRUNCATED AT 250 WORDS)