A cytokine-inducible extrahepatic human indoleamine 2,3-dioxygenase ( h IDO1) catalyzes the first step of the kynurenine pathway. Immunosuppressive activity of h IDO1 in tumor cells weakens host T-cell immunity, contributing to the progression of cancer. Here we report on enzyme kinetics and catalytic mechanism of h IDO1, studied at varied levels of dioxygen (O 2 ) and l -tryptophan ( l -Trp). Using a cytochrome b 5 -based activating system, we measured the initial rates of O 2 decay with a Clark-type oxygen electrode at physiologically-relevant levels of both substrates. Kinetics was also studied in the presence of two substrate analogs: 1-methyl- l -tryptophan and norharmane. Quantitative analysis supports a steady-state rather than a rapid equilibrium kinetic mechanism, where the rates of individual pathways, leading to a ternary complex, are significantly different, and the overall rate of catalysis depends on contributions of both routes. One path, where O 2 binds to ferrous h IDO1 first, is faster than the second route, which starts with the binding of l -Trp. However, l -Trp complexation with free ferrous h IDO1 is more rapid than that of O 2 . As the level of l -Trp increases, the slower route becomes a significant contributor to the overall rate, resulting in observed substrate inhibition. [ABSTRACT FROM AUTHOR]