Asp 274 and His 346 Are Essential for Heme Binding and Catalytic Function of Human Indoleamine 2,3-Dioxygenase*

L -Tryptophan is the least abundant essential amino acid in humans. Indoleamine 2,3-dioxgyenase (IDO) is a cytosolic heme protein which, together with the hepatic enzyme tryptophan 2,3-dioxygenase, catalyzes the first and rate-limiting step in the major pathway of tryptophan metabolism, the kynurenine pathway. The physio-logical role of IDO is not fully understood but is of great interest, because IDO is widely distributed in human tissues, can be up-regulated via cytokines such as inter-feron- (cid:1) , and can thereby modulate the levels of tryptophan, which is vital for cell growth. To identify which amino acid residues are important in substrate or heme binding in IDO, site-directed mutagenesis of conserved residues in the IDO gene was undertaken. Because it had been proposed that a histidine residue might be the proximal heme ligand in IDO, mutation to alanine of the three highly conserved histidines His 16 , His 303 , and His 346 was conducted. Of these, only His 346 was shown to be essential for heme binding, indicating that this histidine residue may be the proximal ligand and suggesting that neither His 303 nor His 16 act as the proximal ligand. Site-directed mutagenesis of Asp 274 also compromised the ability of IDO to bind heme. This observation indicates