The Endogenous Metabolite Hypochlorite Activates Indoleamine 2,3-Dioxygenase-1 for Catalysis: Functional and...

Indolamine 2,3-dioxygenase (IDO1) catalyzes the oxidative cleavage of l-tryptophan (l-Trp) to N'-formyl-l-kynurenine (l-NFK). The generally used in vitro assay for probing IDO1 activity relies on ascorbic acid and synthetic methylene blue to initiate the reaction by ferric iron reduction, with the endogenous activator still uncertain despite several reductants being proposed. Here we demonstrate sodium hypochlorite, commonly known as bleach, functions as an oxidative activator, bypassing the need for a reductant. Other hypohalous acids, generated in situ by lactoperoxidase (LPO) or in aqueous solutions of I₂ or Br₂, also activate rhIDO1. Importantly, complete conversion to l-NFK occurs with sub-stoichiometric hypohalous acid relative to l-Trp with total turnover ≥ 2000. The overall reaction is demonstrated to remain O₂-dependent with hypohalous acids. Steady state kinetic analysis with different oxidative activators revealed typical Michaelis-Menten kinetics with respect to l-Trp without substrate inhibition, which contrasts past analyses. Finally, 1-methyl-l-tryptophan, a reported inhibitor and poor substrate for rhIDO1, was reexamined with the hypohalous acid-dependent conditions revealing it's a substrate with improved catalytic efficiency compared to l-Trp. Along with this unanticipated result, the in vivo functional and mechanistic implications of the discovered hypohalous acid-dependent IDO1 activity are discussed.