The oxygenated form of L-tryptophan 2,3-dioxygenase as reaction intermediate.

In order to clarify the reaction mechanism of Mryptophan 2,3-dioxygenase (L-tryptophan: oxygen oxidoreductase, EC 1.13.1.12), a hemoprotein, spectral and kinetic studies were carried out with highly puritied enzyme preparations from Pseudomonas fluorescens (ATCC 11299). A new spectrally distinct species of the enzyme heme omx; 418,545, and 580 mp) was observed during the steady state of the catalytic reaction. The formation of the new spectral species was absolutely dependent on the simultaneous presence of both oxygen and L-tryptophan with the ferrous enzyme. In the absence of L-tryptophan, the ferrous heme in the enzyme was oxidized to a ferric state by molecular oxygen. Available evidence indicated that the observed spectrum was due to a ternary complex of oxygen, the enzyme, and L-tryptophan and represented the oxygenated form. By means of rapid reaction spectrophotometry, the oxygenated form was shown to be an obligatory intermediate of the reaction. The rate of the over-all reaction, as judged by the accumulation of L-formylkynurenine, was always proportional to the amount of the oxygenated form present during the entire course of the reaction. The rate constant for the decomposition of the oxygenated form was estimated to be 19 set-I, which agreed well with the turnover number of the enzyme (18 set-l). The rate constant for the binding of oxygen with the ferrous enzyme in the presence of L-tryptophan and that for the reverse reaction were also determined to be 5 x lo6 M-I set-l and 230 set-I, respectively. Thus, the binding of oxygen with the ferrous enzyme in the presence of L-tryptophan is a reversible process, as are those with hemoglobin and myoglobin. Some properties of the Pseudomonas enzyme are also described.