The initial step in the l-kynurenine pathway is oxidation of l-tryptophan to N-formylkynurenine and is catalyzed by one of two heme enzymes, tryptophan 2,3-dioxygenase (TDO) or indoleamine 2,3-dioxygenase (IDO). Here, we address the role of the conserved active site Ser167 residue in human IDO (S167A and S167H variants), which is replaced with a histidine in other mammalian and bacterial TDO enzymes. Our kinetic and spectroscopic data for S167A indicate that this residue is not essential for O 2 or substrate binding, and we propose that hydrogen bond stabilization of the catalytic ferrous-oxy complex involves active site water molecules in IDO. The data for S167H show that the ferrous-oxy complex is dramatically destabilized in this variant, which is similar to the behavior observed in human TDO [Basran et al. (2008) Biochemistry 47, 4752-4760], and that this destabilization essentially destroys catalytic activity. New kinetic data for the wild-type enzyme also identify the ternary [enzyme-O 2-substrate] complex. The data reveal significant differences between the IDO and TDO enzymes, and the implications of these results are discussed in terms of our current understanding of IDO and TDO catalysis.