Indoleamine 2,3-dioxygenase. Equilibrium studies of the tryptophan binding to the ferric, ferrous, and CO-bound enzymes.

The equilibrium constants for the tryptophan binding to indoleamine 2,3-dioxygenase, a protohemoprotein, in its ferric (Fe3+), ferrous (Fez+), and CO-bound (Fea+CO) forms were determined by the spectrophotometric titration method. All these enzyme derivatives showed marked spectral changes upon tryptophan binding. Such a spectral change for the ferric enzyme was, however, specific to the L isomer and accompanied the spin state change of the ferric heme from high spin to low spin. The dissociation constants (Kd) thus obtained for the complexes of L-tryptophan with the ferric, CO-bound, and ferrous enzymes in 0.1 M potassium phosphate buffer, pH 7.0 at 24OC, were 5.8 111~, 350 pM, and 13 ELM, respectively, being in a ratio of 450:27:1. The Kd for the ferric enzyme was about 450 times greater than the K,,, and agreed well with the substrate inhibition constant (Ki), indicating that L-tryptophan binding to the ferric enzyme is not catalytically significant, but is directly correlated to the substrate inhibition which is stereospecific to the L isomer. The Kd for the ferrous enzyme showed a good agreement with the Km at various pH values examined. Assuming that the CO-bound enzyme (Fe”C0) has a tryptophan-binding property similar to the oxygenated enzyme (Fe2+02 or Fe3+02-), it is suggested that the binding of tryptophan to the ferrous enzyme ( K d Km) rather than that of tryptophan to the oxygenated enzyme (& # K,) is involved in a steady state of the catalytic reaction. Available evidence described above provides further support to the following reaction mechanism, in the principal pathway, the ferrous enzyme binds predominantly with tryptophan, followed by molecular oxygen. The ferric enzyme, on the other hand, binds first with the superoxide anion and the resulting oxygenated enzyme reacts with tryptophan either directly or via the ferrous enzyme to form the ternary complex. The ferric enzyme. tryptophan complex appears catalytically inactive.