l-Tryptophan 2,3-dioxygenase (TDO) is a protoheme-containing enzyme that catalyzes the production of N-formylkynurenine by inserting O2 into the pyrrole ring of l-tryptophan. Although a ferrous–oxy form (Fe2+–O2) has been established to be an obligate intermediate in the reaction, details of the ring opening reaction remain elusive. In this study, the O2 insertion reaction catalyzed by Pseudomonas TDO (PaTDO) was examined using a heme-modification approach, which allowed us to draw a quantitative correlation between the inductive electronic effects of the heme substituents and the substituent-induced changes in the functional behaviors of the ferrous–oxy form. We succeeded in preparing reconstituted PaTDO with synthetic hemes, which were different with respect to the inductive electron-withdrawing nature of the heme substituents at positions 2 and 4. An increase in the electron-withdrawing power of the heme substituents elevated the redox potential of reconstituted PaTDO, showing that the stronger the electron-withdrawing ability of the heme substituents, the lower the electron density on the heme iron. The decrease in the electron density of the heme iron resulted in a higher frequency shift of the C–O stretch of the heme-bound CO and enhanced the dissociation of O2 from the ferrous–oxy intermediate. This result was interpreted as being due to weaker π back-donation from the heme iron to the bound CO or O2. More importantly, the reaction rates of the ferrous–oxy intermediate to oxidize l-Trp were increased with the electron-withdrawing ability of the heme substituents, implying that the more electron-deficient ferrous–oxy heme is favored for the PaTDO-catalyzed oxygenation. On the basis of these results, we propose that the initial step of the dioxygen activation by PaTDO is a direct electrophilic addition of the heme-bound O2 to the indole ring of l-Trp.