A cytokine-inducible extrahepatic human indoleamine 2,3-dioxygenase, isoform-1 (hIDO1) catalyzes the first step of the kynurenine pathway. This catalytic depletion of L-tryptophan (L-Trp) results in a weakened T-cell immunity of the host, driving progression of cancer. Here we report on steady-state and single-turnover kinetics of hIDO1, aimed at establishing the nature of the catalytically active metal-oxygen species in this enzyme. We monitor initial rates of O2 consumption on a Clark-type oxygen electrode to determine the parameters for enzymatic turnover at physiologically-relevant levels of dioxygen (O2) and L-Trp. Using a cytochrome b5-based activating system, we quantify the effects of O2 concentration on catalysis, supporting a steady-state rather than a rapid equilibrium kinetic mechanism, where the rates of individual pathways (leading to the ternary complex) are significantly different, and the overall rate of catalysis is dependent on contributions of both routes. Additionally, we use a peroxide, meta-chloroperoxybenzoic acid (mCPBA), to cleanly generate ferryl species in hIDO1, and monitor their formation and decay by stopped-flow spectrophotometry. We report on formation of a very short-lived ferryl radical-cation intermediate (Cpd I) from ferric hIDO1 and characterize its fast conversion to a neutral ferryl species (Cpd II). Formation of Cpd II occurs simultaneously with intraprotein electron transfer and generation of an enzyme-centered radical, observable by cryogenic X-band EPR. Oxidation of ferrous hIDO1 by mCPBA, employing anaerobic techniques, allows for comparison of rates of Cpd II reduction in intact and singly-oxidized hIDO1. We discuss the role of ferryl derivatives in hIDO1 catalysis of L-Trp dioxygenation, contrasting our results with those in the published literature, where a neutral ferryl species was proposed as a catalytically active intermediate in dioxygenation of L-Trp by hIDO1.

Dioxygen activation by human indoleamine 2,3-dioxygenase, isoform-1 (hIDO1): The role of ferryl derivatives in catalysis

254th National Meeting (2017)

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